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XF24 Manual

1. a oO 60 70 s0 30 40 50 TIME min 62 A eahorse Bioscience OCR ECAR By using the Y2 axis for ECAR we can now put all the kinetic data on a single graph filled circles for OCR filled squares for ECAR however the graph is more difficult to interpret NEURONAL BEP 090708 HEURONALBEP090708 A2_782009_1416 By using the Group Feature along the X axis the data is displayed by rate measurement One loses the kinetic nature of the data but gains the ability to look at both OCR and ECAR responses together in an organized and easy to read bar graph format OCR pMoles min uw Hdw yvo3 CTL 25 2 CTL 25 4 CTL 25 0 OFR 25 2 OFR 25 1 OFR 25 0 GROUP One may also wish to plot OCR vs ECAR PPR which allows individual rate measurements to selected deselected In the example rates 4 and 6 9 and 12 are displayed to reflect the OCR and ECAR rate data immediately before and immediately after injections respectively Note that this format shows the shift from basal rates to enhanced diminished rates after injection and provides information as to how the aerobic and glycolytic pathways of the cell respond to compounds that alter the metabolism of the cell E amp oc O OCR vs ECAR 63 Aeahorse Bioscience D Advanced Analysis Viewing and interpreting the Level Data One can probe deeper into the data by using the various graphing options to display data in accor
2. Group 2 O gt F gt R Oligomycin gt FCCP gt Rotenone Column and Group assignments Well Grid Layout 5 Compound Preparation Prepare the following compounds in XF DMEM Assay media as described in Appendix VI 10 ug ml Oligomycin 3 0 uM FCCP and 1 0 uM Rotenone 6 Media Change and Cell Preparation a Remove growth medium from each well leaving 50 ul of media b Wash cells with 1000 ul of pre warmed DMEM running media remove 1000 ul as above and add 625 ul of DMEM running media 675 ul final c Incubate in 37 C incubator without CO for 60 minutes to allow cells to pre equilibrate with the assay medium 81 Deahorse Bioscience 6 Loading Sensor Cartridge Warm compounds to 37 C prior to loading sensor cartridge and load the compounds into the injector ports as follows Columns 1 2 Load 75 83 and 93 ul of XF Assay media DMEM into ports A B and C respectively Columns 3 6 Load 75 ul of 10 ug ml Oligomycin into ports A Load 83 ul of 3 0 uM FCCP into ports B Load 93 ul of 1 0 uM Rotenone into ports C 8 Protocol Commands Command Time min Port Calibrate Eouilbrate Mix LoopEnd e Measure 3 J E E Loop Start 2X J Mix s Wat o 2 J Measure 3 J __Iniect_ ___ Loop Start 2X J Mix 3 Wait 2 Measure 3 J p En 9 Results and Discussion This assay is derived from the classic experiment to probe mitochondrial function and serves
3. Instrument Setup Database Screen Aeahorse Bioscience Administration These are settings and subsystems typically used by Seahorse Bioscience for instrument set up Hardware settings are fixed by Seahorse Bioscience and cannot be accessed without a password The Subsystem Buttons allow users to modify certain hardware and software settings Instrument ID displays the serial number of the instrument Communication There are three buttons indicating the communication status of the controller with the instrument barcode reader and PS if used All buttons should be green This indicates that the controller is communicating properly with the other components of the system The Calibration Screen should look like the screen on the right The settings are set by Seahorse Bioscience for optimal instrument and consumable performance Changing these default settings may affect the quality of the sensor cartridge calibration The Parameters button is only used for troubleshooting calibrations an tector Instrument Setup Administration Screen derrr ipieg torpe ms Gormmurecaion Settings mairumeri r F a Ey z Cimma sellings Barcode aadar omm RY Commun caton settings Plate Prap Subsystems Communication Cabbration setings fst reer Calibration plate is in Calibrate position cane m Covwery Sartrtge i leaded on tap of Cabra h Wait aker loadin
4. 141 i Aeahorse Bioscience Copying Saving and Exporting Graph and Well Grid Images Several options are available via the popup menus that appear when right clicking on the well grid and graph objects Both allow copying of the image that can be directly pasted into any Word Excel or Powerpoint document In addition both menus have a Save option that supports saving a snapshot of the object to an image file The standard copy option yields a graph without a grey border while the enhanced copy option yields a graph with a grey border See below for saving a high resolution image of a graph To save an image of the well grid 1 Right click on the well grid and select Save Grid from the popup menu The Save As dialog will appear as shown in the figure to the right Enter in a filename and choose a file type TIFF JPEG and BMP formats are supported lf additional pixel resolution is required in the image click on the checkbox to Use enhanced mode for the save The saved image will be 3 times larger than what is displayed Save As Er My Computer fd My Documents G3 Shortcut to My Assay Template B My Documents W My Computer My Network Places 9 Desktop Icons Misc Training Data k File name Save astype TIFF File tf Cancel iW Use enhance mode for the save Image will be 3 bigger Save As dialog for the Well grid 142 Aeahorse Bioscience To save an i
5. Modifying Instrument Settings The new recommended values for the ECAR calculation settings are automatically set after installing and running the new software XF Reader software version 1 4 and higher They will be the default values for future assays Modifying Existing Data Files The following procedure shows how to apply the new ECAR calculation parameters to your existing data files 106 Aeahorse Bioscience 1 In the Files screen browse for and load an XLS or XFD data file display XFD files by Do background correction changing the file display filter in the lower left corner Eam settings of the Files screen After loading the file go to the Buffer Capacity Volume Results screen to confirm the data was loaded Settings for pecan o e v 2 Inthe Run screen select Ev the Settings tab in the left rs hand panel see Figure SFP pe 21 3 Display the settings for ECAR Modify the Technique and Edge Offset Plate Compound Desc Protocol Info New Settings for ECAR The ECAR data is automatically recalculated Go to the Results screen to view the data To save the data go to the Files screen and click on Save As to generate a new data file Proton Production Rate PPR Calculations and Settings Proton Production Rate PPR was introduced as an alternative measurement of acidification to Extra Cellular Acidification Rate ECAR Unlike ECAR PPR accounts for variations in buffe
6. Setting the Threshold Displaying the Threshold To view the thresholded data inthe graph sean vy and well grid BaseLine Options Time Zero Options 1 Click on the Display threshold Threshold Options 2 Turn on the Display th reshold Link Y1 display variable Do Background Correction option as shown to the right Advanced Graph Options Close 3 Close the graph settings dialog Enabling Display threshold 4 Click on the Well Group Mode button so it is not outlined in green Note Well group mode is not compatible with the threshold feature 133 Deahorse Bioscience Viewing the Threshold When the threshold is displayed the well grid will become colored blue and red The red wells fall within the threshold and the blue wells fall outside the threshold Scroll through the rates to see how the colors change over the course of the assay Thresholded well grid OCR vs ECAR Avg Rate 5 OOCR 1000 hi T K 400 43 77 111 145 180 ECAR mpHi min When graphing an analyte versus another analyte the threshold region is indicated by an ellipse The ellipse is only an approximation The actual threshold region is the rectangle that bounds the ellipse OCR pMoles min Threshold when graphing an analyte vs another analyte OCR vs TIME Avg When graphing an analyte versus time the threshold region is indicated by a solid red line bounded by two dotted red lines i
7. The XF Assay Wizard and Creating XF Assay Templates The Assay Wizard is used for creating an XF experimental template This may be performed on the instrument or on a PC but must be done using the XF Reader software The different sections of the wizard can be accessed at any time using the tabs Each section may be broken down into one or more sub sections that can be selected via buttons across the top of the section Navigation of the wizard can also be performed using the Next and Back buttons at the bottom These buttons will take you through the wizard in sequential order When starting the Assay Wizard the following should appear You may open a previously constructed template using the Load Template button Cole Media APPR Cempounds Beck Ceitection Oroupsi labels Protoced End and modify the information in the fields as desired or you may choose to begin with an empty template by choosing the Clear Assay button The Project Information Project Mame Project Number Principal investigated Load template button will access the template directory specified in the instrument set up When using the Wizard a reset button is available in the lower left corner In any given section of the Wizard use the Reset button to clear out that particular section Instrument Operator Deparment Result File Hame XF24 Assay Wizard Screen General Description of the Assay Wizard Tabs and building an XF Assay Template Gene
8. The OCR data is automatically recalculated Go to the Results screen to view the data To save the data go to the Files screen and click on Save As to generate a new data file Do background correction 101 Deahorse Bioscience Algorithm Comparison XF24 ASSAY DATA In this graph of oxygen level data note how the levels drop into the negative range for many of the wells This is an artifact of the assumptions made in the Original algorithm The oxygen data has fallen outside the recommended range a total drop in partial pressure of O2 lt 50 mmHg FAO XF24 ASSAY DATA 1 xls After applying the new algorithm note the change in the oxygen level data The Y axis scale has been adjusted 50 TIME min Oxygen Level Data Calculated with New Algorithm XF24 ASSAY DATA Now review the OCR data Here are the rates using the original algorithm Nn 2 i m Oo 34 50 TIME min 102 Aeahorse Bioscience Original OCR Data XF24 ASSAY DATA After applying the new algorithm note that the higher rates have decreased The baseline rates have not changed appreciably since they were already within the recommend range n 2 m O Here is another example of oxygen level data based on the original algorithm 20 2 3 4 TIME min The new algorithm has been applied Note that the level data after the injection from Port B no longer falls into the negati
9. b Aeahorse Bioscience XF24 Extracellular Flux Analyzer and Prep Station Installation and Operation Manual Software Version 1 7 100736 400__MANUAL XF24 W PREP STATION_RB Deahorse Bioscience Revision history REV A RELEASED PER ECO 161 02 11 10 REV_B RELEASED PER ECO 212 01 12 11 Copyright Copyright 2009 Seahorse Bioscience Inc All rights reserved Trademark Information Microsoft Windows Excel and Vista are registered trademarks of Microsoft Corporation All other trade names are properties of their respective companies Seahorse Bioscience Inc 16 Esquire Road N Billerica MA 01862 800 671 0633 978 671 1611 Fax Northwest Research Center 16000 Mill Creek Blvd Suite 205 Mill Creek WA 98012 425 338 4703 Submit technical support questions or comments about our documentation to support seahorsebio com For the latest information on products visit our web site at www seahorsebio com Printed in U S A Aeahorse Bioscience General Assay Flow Chart for all XF Assays XF Sensor seed cells at proper Prepare stock Cartndge density on XF24 TC plate Prepare XF compounds Hydration Assay Media to be injected Day Prior to Assay materials preparation Remove growth media 1X XF Assay Wash cells with 1X Hedium pH 7 4 XF Assay medium 37 C Thaw and add XF Assay media wam to 37 C to proper volume manually or use PS Incubate 37 C 60 min Add stock solution 5 with Initial ass
10. 1 0 M 2 deoxyglucose 2 DG For a 1 0 M 2 DG solution combine 0 492 g 2 DG Sigma D6134 in 2 7 ml DMEM or KHB XF Assay media Check volume and bring to 3 0 ml final pH to 7 4 using 4 8 ul of 0 1 N NaOH Check with a narrow band pH strip Hint compare the color of 2 DG solution to the same volume of unbuffered DMEM media to accurately pH Make sure both solutions are at the same temperature during comparison You may also test with a narrow band pH strip 158 at S ri s i EI eahorse Bi oscience Bulk Preparation of Palmitate BSA Complex Materials Sodium palmitate Sigma P9767 mw 278 41 Ultra fatty acid free BSA Roche 03 117405 001 mw 68 000 Tissue culture grade deionized water Gibco 1523 0147 5M NaCl solution Sigma S6316 Glass beaker 1 L 2 Glass beakers 250 mL 2 Glass Erlenmeyer flask 50 mL 1 Small stir bars 3 Heated stir plate 2 Thermometer 2 150 mL filter unit 0 22 micron Corning 430626 Glass vials 4 mL National Scientific B7999 2 Ice Il Preparation of 1 mM Sodium Palmitate 0 17 mM BSA Solution 6 1 FA BSA Note keeping each solution at the specified temperature is critical in this protocol A Setup prior to BSA and sodium palmitate solution preparation 1 Warm about 200 mL tap water in each of 2 1L beakers in 37 C water bath 2 Warm 250 mL beaker with stir bar in 37 C incubator 3 Make 300 mL 150 mM NaCl by adding 9 mL 5 M stock to 291 mL dH2O B BSA solution preparation
11. 131 5 eahorse Bios cience 4 The labels are FETS initially displayed in the upper left corner of the graph Click and drag labels to the desired locations OCR pMoles min Note When working with the data file in Excel the labels are saved with the data fi le But labels created i 10 4 20 8 31 2 41 6 52 0 62 4 T28 23 2 93 6 104 0 in the XFReader24 TIME min software will not be saved to the data file Label displayed on the Graph Data Threshold Option Overview When viewing data in non well group OCR vs ECAR Avg mode a new thresholding option is Rate 5 now available OCR 1000 are 600 A target value and range are specified and all data points are sorted based on this threshold Data outside the threshold are colored blue and those points inside the threshold are colored red 400 p a oc O The figure to the right shows an example graph with a threshold 43 77 111 applied ECAR mpH min Threshold applied to scatter plot of OCR vs ECAR 132 Peahorse Bioscience Setting the Threshold j To set the threshold Minimum Value 1 Click on the Maximum Value 2 For each axis of interest set the PAAL Threshold and Threshold Num Decimals range fields as shown in the figure to the right okie Threshold range t 3 Close the graph settings dialog Show Error Bar Note a threshold cannot be defined for the x axis when it is set to TIME
12. 16952 23 2179 89 3914 89 1548 18 0 00 0 00 0 00 0 00 0 00 0 00 859 93 600 48 165 33 1375 21 1007 61 i 1281 40 1900 77 1234 19 449 39 2780 56 2338 30 f 3219 43 2205 03 1624 06 597 25 2969 65 2787 70 i 4313 07 2097 98 1936 73 641 42 f 2720 45 2929 57 4779 64 1881 37 1720 26 700 69 2220 01 2973 72 5087 91 1505 57 1436 79 585 35 160 2109 21 2855 40 3 5359 30 4 3 3 10450 66 8552 51 3139 43 14175 09 14892 31 24040 75 SDev SDev SDev 0 00 0 00 f 0 00 0 00 430 38 1036 77 f 1171 67 l 790 83 936 47 l 2288 41 2941 89 i 1928 14 1139 08 i 2685 22 3931 27 i 2392 16 1189 65 2677 56 l 4402 03 2269 36 1080 63 f 2478 11 4701 51 i 2036 04 841 75 i 2380 92 4940 12 i 1940 52 n n CN IN NY NY Fa ee Re eS WW WIWINIRINIS N n WO NO S CO co I a wiy Oo CO Oo N On Oo BRO O C CO SI lt n f Time Events Calibration AUC Anova ocR1 lt Ready The area data is broken down per well and per rate as well as the average per group and per rate The areas are normalized to the starting rate which is why the starting rate is zero This graphic demonstrates how the AUC is calculated Assuming the third point is the starting rate the yellow region representing the baseline area is subtracted from the total area The remainder the red region is the AUC used in the analysis 119 eahorse Bioscience
13. Bar graph option and graph legend options Many of the features are explained in more detail in Appendices IV and V 58 Deahorse Bioscience Rate Options Dialogue Button Scatterplot Settings Options Display Selection Display rate with data point Display current rate Opens the Rate Options dialogue which Display selected rates allows the user to specify which rate data is LPP okie a Wen displayed on the graph when the X axis is a Link points with directional lines parameter other than Time Either the Draw injection Marker current rate or selected rates may be displayed It also allows display options Cancel such as labels and linking points with directional lines NEURONAL BEP 090703 NEURONALBEP090708 A2_782009_1416 2 For example if OCR is plotted vs PPR ECAR it may be useful to display only the basal rate data immediately before injection and the perturbed rate data immediately after injection then to link the shifts in OCR and or PPR using directional lines OCR pMoles min Movie Function Button i Opens a window that shows the data in an animated format You may play pause alter the speed etc Movie Function Note that the data viewer and analysis features described above are shown in the Excel version The XF data files may also be opened in the XF reader software In addition to features listed above the Excel data file also i
14. Click on the kw button to open the graph settings dialog 2 Click on the Time Zero Options button 3 As shown in the figure below an injection point or protocol command can be selected as the new time origin Click OK after making a selection 4 Close the graph settings dialog 128 Aeahorse Bioscience First recorded tick Injection point Pota Yv Other point 3 1 Measure for 3 Min 0 sec Time Zero Options In the figure below two graphs are shown with different origins for the time axis The left graph shows the default time origin In the right graph the time origin has been shifted to the beginning of the first measurement OCR vs TIME Avg OCR vs TIME Avg OCR pMoles min OCR pMoles min 0 20 40 60 80 100 TIME min TIME min Graphs with two different time origins Adjusting Axes Interactively in the Data Viewer Graph When analyzing data it s often necessary to manually adjust the min and max values of the graph axes to focus in on a subset of data In the Excel based Data Viewer this can now be done interactively 129 Aeahorse Bioscience 1 As shown in the figure to the right click and drag to create a square region in the graph 2 As shown in the next figure the graph will magnify the defined region by automatically adjusting the Min and Max values of the X Y1 and Y2 if used axes 3 Toreset the graph axes click on the 5 button Note This featu
15. Note 1 2 of the solution is intended for making aliquots for vehicle control in FAO assay The remaining 1 2 is conjugated with sodium palmitate as in section C 1 Weigh out 2 267 g ultra fatty acid free FAF BSA 2 Add to 100 mL 150 mM NaCl in 250 mL glass beaker while stirring in a stir plate 2 Cover beaker with parafilm and place in one of the 1L water baths pre warmed to 37C on a heated stir plate adjust heat as needed to maintain temperature near 37 C but never more than 40 C keep a thermometer in water bath 3 Stir till BSA is completely dissolved 4 Transfer BSA solution to the upper chamber of 150 mL filter unit in laminar flow hood Filter with vacuum 5 Transfer 50 mL BSA to pre warmed 250 mL beaker cover with parafilm return to 37 C water bath and resume stirring 6 Dilute the remaining 50 mL BSA with 50 mL 150 mM NaCl for 0 17 mM BSA stock 7 Aliquot 4 mL glass vial and freeze at 20 C for use as BSA control C Sodium palmitate solution preparation 1 Once BSA is stirring in water bath weigh out 30 6 mg of sodium palmitate and add to 44 ml of 150 mM NaCl solution in 50mL Erlenmeyer flask 2 Cover flask with parafilm and weight with lead ring Place in the other pre warmed 1 0 L water bath on a heated stir plate heat toC Qvhile stirring thermometer in water bath 159 Deahorse Bioscience 3 Palmitate solution will appear increasingly cloudy as temperature reaches 50 60 C but will become clear near
16. O gt F gt R M 98 71147423 3 292815297 13 1 1 09 46 AM 2 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 Temp Corr 0 0 0 1 1 09 46 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 113 5124406 2 7891295 7 2 1 18 14 AM 1 C2012 C2012 C2012 C2012 C2C12 C2C12 C2C12 O gt F gt R M 104 5989241 4 119635143 13 2 1 16 14 AM 2 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 Temp Corr 0 0 0 2 1 18 14 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 114 4429369 3 396044699 7 3 1 26 42 AM 1 C2012 C2012 C2012 C2012 C2012 C2012 C2C12 O gt F gt R M 106 2207208 4 254306767 13 3 1 26 42 AM 2 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 Temp Corr 0 0 0 3 1 26 42 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 116 3342381 3 103431531 7 4 1 35 10 AM 1 C2012 C2012 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 105 1834403 4 173768937 13 4 1 35 10 AM 2 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 Temp Corr 0 0 0 4 1 35 10 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 111 1743351 4 233381697 7 5 1 43 46 AM 1 C2012 C2012 C2012 C2012 C2012 C2012 C2C12 O gt F gt R M 40 37620989 1 677677696 13 5 1 43 45 AM 2 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 Temp Corr 0 D 0 5 1 43 46 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL
17. Plate Manager Allows the user to control the probe and tray positions manually should there be an issue or problem with the instrument SHB may ask you to access these settings should you experience issues with barcode reading stripping the cartridge calibration or similar issues otherwise you will likely not need to use these commands User Manager Allows the user to edit the User List and User Info Please see Appendix V for more information on user preferences After completion of instrument setup click End Instrument Setup Mode iy a ee Sede res eter User Manager Screen Cleaning and Routine Maintenance The XF24 instrument was designed for minimal cleaning and maintenance by the user Note all consumables are disposable and none of the instrument components come into contact with the cell plate at any time preventing cross contamination of biological or chemical E i at tl it materials However there are some minor tasks Pr el HER N hii e y nt oar apd ON 1 that can be performed by the user if necessary w id G j De il Probe Cleaning he lf cell culture plate wells are over filled or over a long period of time the optical surface on the bottom of the measurement probes may require cleaning Use a cotton tipped stick and 95 ethanol to clean these optical windows beneath each probe Take care not to scratch the surface and do not use a cleaner that leaves a residu
18. Plating info Information may be entered about the plating of cells for an assay Cell Layout Allows the user to assign IE specific information about the cells in mra 515 each well Note that one could use b more than one type of cell per plate This is where this info would be assigned To assign information to the wells perform the following steps a Highlight cell type to be used e g TRS C2C12 b Enter the seeding count per well O e g 30000 c Select all wells by pressing the upper left corner box of the plate layout d Deselect individual cells to be used for Background Correction e g Blank wells A1 B4 C3 and D6 e Click on the assign to wells button t h Busi Cran ani Fii fisi Assay Wizard Cells Cell Layout Screen Repeat steps a e to assign Blank to the reference wells Hint simply select the four wells to be used then press assign Be sure to enter a seeding count of 0 here You may use the Zoom Grid button to view the Cell Layout in full screen 86 Aeahorse Bioscience Media and PPR Tab Media Info This tab is for documenting information about the assay running media and functions similarly to the growth media tab above Different assays may use different running media e g mitochondrial studies may use a DMEM while FAO experiments will use a KHB buffer Note that if the Load from Database button is used four 4 pre defined media choic
19. Rate Data Plate view all of the rate data for each analyte is shown in a plate view by measurement Rate Data all of the data for each analyte by column Time Events Lists all of the time events associated with the experiment including Calibration mix wait measure and injection commands Calibration Used to determine if the sensors passed the calibration requirements and provides calibration information and statistics about each individual sensor Well Grid Features and Options Plate Column Select Select Deselect Deselect Button Buttons Row Select Deselect Buttons Group Average Baseline Button Button Group Normalize Edit Button Button NS Global Group Grouping Boxes Button 53 Deahorse Bioscience Individual wells may be selected or deselected by clicking on the appropriate well Columns or rows may be selected or deselected by clicking on appropriate column number or row letter The entire plate all wells may be selected or deselected by clicking on the square in the upper left corner of the plate layout Group Average Button Calculates the mean of the selected wells in each group and displays the mean top number and the error bottom number The error is reported at either SD or SEM user defined These values appear in the Group Boxes below the plate layout Group Boxes display the mean rate value and error associated with each group The Group Boxes may be selected and deselected
20. Technical Specifications Dimensions Width x height x depth Width x height x depth Controller 18 x 22 max x 14 14 x 19 x 20 46 cm x 56cm x 36cm 15 x 19 x 16 38 cm x 48 cm x 41 cm 36cmx 41cm x 51cm Analyzer Controller 20 Ibs 9 Kg 35 Ibs 16 Kg Analyzer 30 Ibs 14 Kg Power 100 120VAC 50 60Hz 220 240VAC Requirements 50 60Hz export 6A 3A Analyzer 1 8A 0 9A Controller Transient Category Il of IEC 60364 4 443 Power cord rating 3 wire grounded AC power cord rated 3 wire grounded AC power 10A or greater cord rated 10A or greater Power fuse ratings 10A 5A in each of Analyzer and Controller Environmental Normal Environmental conditions Normal Environmental Conditions indoor use altitude to 2000 m conditions indoor use Room Temperature 60 86F 15 C to 30 C 60 to 86 F 15 C to 30 C No direct sunlight No direct sunlight Humidity less than 80 for temp up to 31 C decreasing linearly to 50 at 40 C 100 150V AC 50 60Hz 220 240VAC 50 60Hz export Internal Controlled to user selected temperature Incubator and bottle holder Temperature not less than 8 C above ambient temp calibrated and set to 37 C user may change both No CO control No CO control Software OS Windows Vista Windows Vista Data Interface RS232c internal RS232c gt USB 1 cable with TCP IP external separate Serial to USB adaptor 10 horse Biosc
21. oO Graph Options Dialogue Point to Point Rates mode 104 Deahorse Bioscience Using middle point vs point to point rates The choice of displaying the rates as middle point or point to point is based part in the preference of the user and part in the actual rate data Example The graphs below show the same data in middle point mode left and point to point mode right Note that for the first two measurements the middle point and point to point modes provide equivalent information about the rates i e the OCR is relatively Stable throughout the measurement period Thus the middle point accurately represents the rate over the course of the measurement period However for the third measurement period after injection of ADP the 0 25 mM ADP blue changes dramatically over the measurement period decreasing from 400 pmol min to 100 omol min while the 2 0 mM ADP orange OCR remains stable through out the measurement period 400 pmol min Thus the middle point accurately represents the 2 0 mM ADP OCR while 0 25 mM ADP OCR is significantly underestimated ISO MITO IM ML 090729 A_1222 AKOS xls ISO MITO IM ML 090729 A_1222 AKOS xls QOCR OOCR n ADP Oligo F 500 EE 0 25 mM ADP 450 1 2 0 mM ADP 400 350 300 450 400 350 OCR pMoles min he OCR pMoles min N 0 2 4 6 8 10 12 14 146 418 2 22 0 2 4 6 8 10 12 4 6 418 2 22 TIME min TIME min Middle point Rate Display Point to
22. to show an acute response in OCR to exogenous fatty acid injection 74 2 at ty Se Deahorse Bioscience 16 Dose Response Experiment using HepG2 cells In this experiment HepG2 cells are seeded at a given density in Seahorse cell culture plates The basal oxygen consumption OCR and extracellular acidification ECAR rates are measured to establish baseline rates The cells will then be treated with increasing concentrations of either 2 4 Dinitrophenol 2 4 DNP or DNP or Carbonylcyanide p trifluourmethoxyphenylhydrazone FCCP Several wells will serve as the control with running media added as a control compound Both DNP and FCCP are proton ionophores that are mobile ion carriers They are uncoupling agents because they disrupt ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase Complex V This leads to a rapid consumption of energy and oxygen without the generation of ATP In this case both OCR and ECAR will increase OCR due to uncoupling and ECAR as the cells attempt to maintain their energy balance by using glycolysis to generate ATP The results from the assay will then be used to plot OCR or ECAR vs DNP or FCCP to calculate an ECs value for the compound under these experimental conditions 1 Reagents and Materials a DNP Sigma 198501 and or FCCP Biochemika 21857 b DMEM Running Media see Appendix VI c DMS
23. with corresponding rate data displayed or removed from the graph respectively Note that only the selected wells of a group are factored into the mean and error values for the entire group This allows the user to remove outlier values if needed or foc view a select set of wells within a group in Gaar Group Edit Button Brings up a I dialogue that allows eY the user to edit the group names and Group and Color Edit Dialogues Excel color designations after the assay has been performed see diagram to right To edit the group name click on the desired field and re enter the new group name To reassign the wells select the desired group on the left side then select the wells you wish to reassign To change the color of the group click on the colored square adjacent to the group name A color palate dialogue will appear Select the desired color from the existing palate or create a customized color 5 Eee See Global Grouping Button Changes the plate view from individual wells to show grouping of wells see diagram to right Groups are colored and labeled as defined by the user in the assay wizard or via the group edit button The average rates and associated error is displayed and will change as the user scrolls through the rate control bar This is a convenient way to view the group data in the well plate format 54 i Aeahorse Biosc
24. www tripplite com en products model ctm txtModellD 3914 Plug the USB end of the adapter into the computer The installation will now complete Assigning a Communications Port to the Keyspan Driver Once the driver is installed it is necessary to check the communications and if necessary assign a communications port to the device 1 Access the device manager For windows XP go to Start gt Control Panel and click on System Next click the Hardware tab and then click Device Manager For Vista click on the Windows icon then select Control Panel Switch to Classic View then follow the above instructions In the device manager expand the Ports amp LPT tree Next to Keyspan should be COMS8 If not right click on Keyspan and select oroperties 4 Click on Port Settings 5 6 Click OK then close the windows In the COM Port settings drop down select COM port 3 Setting up Communications in the XF Software After driver installations and once the communications port has been set correctly you may need to let the XF software know which port to use to communicate with the Plate Preparation Station oS a eS Start the XF24 software Login to the software Click on Instrument in the lower right corner of the screen Click on Administration on the right side of the screen Click on Communications on the left side of the screen Unde
25. 0 TIME min ea as E LA Deahorse Bioscience Larger changes would indicate a potential problem likely an inadequate amount of time for the sensors to re equilibrate during the mix and wait cycles In such a case the O2 level data may look more similar to the graph to the left bottom Note that before injection the O2 level is returning to ambient levels however after an injection of DNP the starting O2 levels decrease with each successive measurement This is due to the fact that the cells are now consuming oxygen at an increased rate and that the sensors are unable to recover during the mix and wait times An increase of mixing time by 1 2 minutes would be sufficient to alleviate this problem Analysis of the level data for pH is prta LIME similar to that for O2 simply choose OH for the Y axis Again one would expect to see little variation in the Starting point of each measurement see yellow dashed line indicating that the pH sensors have an adequate amount of time during mixing and waiting to re equilibrate to the ambient pH value of the media In _ this example note that the starting pH values for each measurement vary within lt 0 05 pH units well within the sae acceptable noise of the instrument Emig A large increase or decrease gt 0 2 pH units AFTER injections would indicate that the OH of the injected compound is significantly different from the running media Care must be taken to en
26. 0 sec uu 6 1 2 3 4 Measure for 5 Min 0 sec 9 7 Loop End 4 8 inject Port A 9 9 Loop 3 times uu 10 5 6 7 Mix for 3 Min 0 sec 11 Time Delay of 2 Min 0 sec Jit 12 5 6 7 Measure for 5 Min 0 sec 2 13 Loop End ae 14 Inject Port B 2 15 Loop 3 times HE 16 8 9 10 Mix for 3 Min 0 sec 6 17 Time Delay of 2 Min 0 sec HL 18 8 9 10 Measure for 5 Min 0 sec 419 Loop End entered the measurement cycle can be entered A Procram end typical measurement cycle consists of a mix wait and lt 0 gt y measure command This 3 step cycle can be copied and pasted by highlighting the desired commands on the left clicking Copy and then Paste or by using the Loop command as shown in the example entire Loops may be copied and pasted as well Assay Wizard Protocol Screen Example D Note that OCR and ECAR values are typically not stable until the second or third measurement cycle thus it is strongly recommended that 3 4 basal measurements are performed prior to injection of compounds in order to establish the baseline metabolic rates for the experiment 92 Aeahorse Bioscience About Mix Wait and Measure Times The length of Mix Wait and Measure cycles depends on the particular baseline OCR of the cells one is using in the experiment For most purposes the following table provides the appropriate Mix Wait and Measure times Suggested Mix Wait and Measure Times for XF
27. 111 2670098 5 687693866 7 6 1 52 15 AM 1 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 38 05175035 1 692014733 13 6 1 52 15 AM 2 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 Temp Corr D D 0 6 1 52 15 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 109 7542319 4 020860974 7 7 2 00 51 AM 1 C2012 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 185 6205709 12 56286954 13 7 2 00 51 AM 2 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 C2012 Temp Corr 0 0 0 2 00 51 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 110 04215 3 947003202 7 6 2 09 19 AM 1 C2C012 C2012 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 202 5978564 14 2699576 13 8 2 09 19 AM 2 C2C12 C2C12 02C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 Temp Corr 0 D 0 8 2 09 19 AM 12 Temp Cor Temp Cor Temp Cor Temp Car CTL 105 66697 4 982930219 7 9 2 17 56 AM 1 C2C012 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 61 99623691 1 950161507 13 9 2 17 56 AM 2 C2012 C2012 C2012 C2C12 C2C12 C2C12 C2012 C2012 C2012 C2C012 C2012 C2012 C2C12 Temp Corr 0 0 0 9 2 17 56 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor CTL 106 2567651 4 026514854 7 10 2 26 24 AM 1 C2012 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 O gt F gt R M 65 56364642 1 654710897 13 10 2 26 24 AM 2 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 C2C12 Temp Corr 0 0 0 10
28. 13 07 16 11 45 FAD sF 455 47 DATA 1 82 OF a0 16 16 39 hed assay data 2 xls Add Delete Clear List The Data Files tab showing two sets of data Viewing Multiple Data Sets After loading a second set the graph will display the data from both files In the figure below the left graph shows the original data and the right graph includes the data added from another file FAO XF24 ASSAY DATA 1 xIs OCR vs TIME Avg OOCR P 1000 eR i Ha E E af perce 600 IE A ve sa GASE i ji FSG zs 200 a ip 80 100 TIME min emt Graphs showing data from one file and from two files To toggle a set of graphs one and off use the checkboxes shown to the left of each file listing in the Data Files tab If only one is selected then the graph title will default to the name of the source file for that data If multiple listings are selected then the graph title will default to a generic indication of the graph content The well grid can only show data from one data set at a time To switch between data sets click and highlight the desired listing on the Data Files tab Continue to use the controls on the Grid amp Graph Controls tab to select which analyte to display in the grid 136 Deahorse Bioscience The figure below shows the Data Files tab with the checkboxes and highlighted listings in use In this example the graph would show the data from the second data set and th
29. 150 ul of growth media to each well bringing the total volume of media in the well to 250 ul When adding media to the wells add it slowly to the sides as to not disturb the newly attached cells E Allow the cells to grow overnight 37 C 5 10 CO3 43 at aia A LA Deahorse Bioscience 7 Preparing Sensor Cartridge for the XF24 Assay For optimum data quality and consistency Seahorse Bioscience recommends hydrating XF24 Sensor Cartridges overnight using the following procedure A B Add 1 0 ml of Seahorse Bioscience XF24 Calibrant pH 7 4 to each well of a seahorse Bioscience 24 well plate Place sensor cartridge on top of plate and store at 37 C without COs overnight or for up to 72 hours If cartridge is being hydrated for more than 24 hours wrap parafilm around the edges to prevent evaporation C Turn on instrument and start XF software to allow instrument to stabilize at 37 C XF Assay Day 2 8 Preparing Cell Plate for XF24 Assay A B C Warm XF Assay media unbuffered DMEM RPMI KHB or other assay media to 37 C Inspect cells in the XF24 cell culture plate under microscope to assure confluence and even seeding Using a multi channel pipet every other tip remove 150 200 ul growth media from each well taking care not to touch pipet tip to the bottom of the wells Leave approximately 50 100 ul of growth media behind in each well Rinse wells by adding 1 ml of warm XF assay m
30. 2 26 24 AM 12 Temp Cor Temp Cor Temp Cor Temp Cor Note that statistical information will be listed only for wells and analytes that are selected This feature is useful for reviewing the statistics associated with the entire experiment and may be useful for identifying trends and troubleshooting the experiment These values may also be copied to other graphing programs if desired AUC Analysis using Multiple t Tests A tool button exists in the data file viewer for AUC Area Under the Curve analysis This is also located under the Special Operations Tab in the Excel Data Viewer Similar to the Group Statistics tool the AUC tool will add a worksheet to the file and perform its analysis based on your well groupings In addition to calculating the area under the curve for the specified analyte t tests are performed to compare the groups and a table of P values is generated 112 Aeahorse Bioscience AUC Analysis Excel configuration _ k E Microsoft Excel Book1 J Ari 4 7 Spelling p The AUC tool uses Exce s Analysis ee ToolPak VBA add in Before you can S use the tool you must first enable the S add in If this feature was not included in e the original installation of Excel you will n Formula Auditing be prompted to insert the Office installation CD Macro AutoCorrect Options Customize Options To enable the add in go to the Jools ee menu and select Add ns Add
31. 70 C D Conjugating Palmitate and BSA 1 Remove parafilm from both beaker and flask 2 Transfer 40 ml hot palmitate solution to the 50 ml of BSA solution stirring at 37 C Palmitate will precipitate if it is allowed to sit in a pipette transfer 5 ml at a time 8 times in a 10 ml pipette taking up and expelling quickly 3 Re cover beaker with parafilm 4 Stir at 37C for 1 hour monitoring temperature of water ba th to keep between 35 and 40 C Add ice to water bath to lower temperature if it reaches 40 C 5 Measure volume in 100 mi glass graduated cylinder and adjust final volume to 100 ml with 150 mM NaCl for 1 mM palmitate stock 6 Adjust pH to 7 4 with 5 10 uL 1 N NaOH 7 Aliquot 4 ml or smaller volume if preferred per glass vial and store at 20 C Ill Thawing Palmitate and BSA for FAO assay A Thaw palmitate and BSA in a 37 C water bath for 10 minutes before loading into the XF cartridge Suggestion if palmitate is thawed immediately before pre incubating cell plate it will be ready to load by the time the medium change is accomplished B Palmitate and BSA are known to be stable at 20 C for at least two weeks and thought to be stable for up to one month The key aspect of successful preparation of the Palmitate BSA complex is to monitor the time and temperature of each step carefully and accurately Notes Specifications of Solutions Palmitate is 1 mM for a 5x injection to 200 uM final It is complexed in
32. 9 a 15 amp am 0 e x 50 O O 25 O 25 aS aS 0 0 2 1 0 1 2 3 4 3 2 1 0 log DNP in uM units log FCCP in uM units Note The ECs curves may be generated using any standard graphing program i e this is not supported in the XF software In these examples Prism5 by Graphpad San Diego CA was used Qualitatively one should expect similar responses as those shown above when using HepG2 cells Also note that the curves above show that at higher doses of uncoupler the OCR actually decreases This is a well documented phenomena indicating that the cells are now less viable due to perturbation of ion flow across all cellular membranes Thus when calculating the ECso it is wise to omit the points at which the OCR value begins to decrease at higher uncoupler the last point in the examples above Please see more information on maximal uncoupler response in the next section 79 2 at ty Se Deahorse Bioscience 17 Bioenergetic Profile Experiment using C2C12 myoblast cells In this experiment C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates The basal oxygen consumption OCR and extracellular acidification ECAR rates are measured to establish baseline rates The cells are then metabolically perturbed by addition of three different compounds in succession that shift the bioenergetic profile of the cell One group will serve as the control with running media added as co
33. Expected Results and discussion As discussed above addition of palmitate to C2C12 cells has a predictable effect on the bioenergetic state of the cells Note that injection of palmitate results in an increase in OCR while the ECAR is relatively unaffected the decrease in ECAR during the time course of the assay is likely due to the shift from 25 mM glucose in the growth media to 2 5 mM glucose in the KHB buffer employed for the FAO assay Typical results are presented in the OCR vs time and ECAR vs time graphs below 73 OCR vs TIME Avg ECAR vs TIME Avg ECAR mpH min a oO o A 4 123 0 00 3 69 17 37 26 06 34 75 4343 52 12 6080 6949 78 18 56 86 8 69 dsm elac Sato aca eke te 6G Golda m ccs TIME min TIME min OCR vs Time ECAR vs Time Qualitatively one should expect similar responses as those shown above when using C2C12 cells It is important to note that absolute values of OCR and ECAR may vary due to technical proficiency what type of and how many cells were seeded the degree of confluence at time of assay whether a consistent monolayer was obtained the exact OH of solutions and added compounds etc Further it should be noted that not all cell lines or cell types may not respond to exogenous fatty acids palmitate E g HepG2 cells appear to have little to no response in an FAO assay Further other cell tyoes may require additional components e g insulin metformin etc
34. Labels The labels for the views can be changed by clicking on the Rename button Enter the new name in the dialog and click OK JYoJH9O Ydd XYY J YO View Tabs with Custom Labels Configuring and Graphing Multiple Views After creating the views click on each tab and customize each view by configuring the Data Viewer In this example the graph in each view has a different set of data and the views are labeled accordingly Click on the Graph tab to see a display of each graph from the four views In this mode the tabs include a button for showing and hiding the corresponding graph So one two or three graphs can be displayed instead of all four 139 rm j FSAI ACEA AA e O i f f J p F eanorseDioscience Microsoft Excel XF24 ASSAY DATA 1 xls i JEJ amp 9 File Edit View Jnsert Format Tools Data Window Help Type a question for help X RET EEN E E E E E E E O aeaa MERIEN E Reply with Changes End Review E X f ee eS a ee ae E Dual Grid Graph yYVvos yoo Hdd HYoJHI0 Jelek A iw w N ho ab ab ab ad ab ab b ab ab j w N N N N ojo N on e N gt a a v se Data Viewer Assay Configuration Level Data Rate Data Plate View Rate Data Calibration gt NUM pas m w a lt Graphs Displayed from each View Saving and Exporting Graph Data The graph s right click context menu contains a new item call
35. a 6 to 1 molar ratio with BSA which is 0 17 mM for a 5x injection to 33 3 uM final The conjugated palmitate can be diluted to a lower concentration with NaCl or KHB This protocol can be adapted to make a 2 mM palmitate solution for 10x injection or 8x etc as desired 160 Peahorse Bioscience Appendix VII Coating of XF Cell Culture Plates The XF cell culture plates are similar to any typical cell culture plate with respect to plasma treatment and cell adherence If cell types are used that need a type of coating the XF plates may be coated with any typical coating including poly D lysine collagen fibronectin and Cell Tak Please follow the respective manufacturer s instructions for coating procedures Appendix VIII Troubleshooting Please visit www seahorsebio com for FAQs For further information please contact your local Seahorse Bioscience Field Application scientist You may also contact Seahorse Bioscience Support via email or phone see below 161 A A 73 r X A gt gt BS eahorse Bioscience Appendix IX Training Completion Check Questions 1 Label the Injection Ports below 1 Port 2 Port 3 Port 4 Port 2 Identify the wells used for Temperature Correction below Temperature Correction Wells used for temperature correction Do temperature correction OK Cancel 162 gt Deahorse Bioscience 3 True or False A typic
36. address 19 When the XF24 is set up in YOUR lab perform a baseline cell run with an acute drug response 2 4 DNP CCCP etc following the guidelines detailed in this protocol Sample templates may be seen in Sections X XIII Send the data file to Seahorse Bioscience to ensure proper function and set up of your new instrument Appendix X Contact Support and Ordering Information For questions about XF technology the XF24 instrument Prep Station XF experimental design data analysis troubleshooting and other information Email support seahorsebio com Phone USA 800 671 0633 option 3 Phone EU 1 978 671 1600 option 3 For ordering XF24 cell culture plates XF24 sensor cartridges and calibrant solution Email orders seahorsebio com Phone 978 671 1600 option 2 Fax Purchase Orders to 978 671 1611 164
37. assays OCR Mix Time Wait Measure pmol min Time 25 50 2 min 2 min 5 min 50 100 2 min 2 min 4 min 100 500 3 2 3 500 1000 4 0 3 1000 Call Support For baseline OCRs above 800 pmol min we suggest you contact a Seahorse Bioscience representative for further information and choosing appropriate cycle times When working with a new or unknown cell line begin with a 3 minute mix 2 minute wait and 3 minute measure cycle Display Tab In the Display tab the user has the option of choosing different ways to display the graph data when the XF run is through Some of the important condiserations for this are font sizes for graph displays line and point sizes and the option to show the legend In Sc the Data button the user can choose f Pie a default graph options including what to show on the X and Y axis as well as what data is used to make error bars standard deviation or standard error of the mean 2 wf FY OS gt bo o E Assay Wizard Display Screen 93 Aeahorse Bioscience End Tab Ends the Assay Wizard The information entered is applied as the Current File and the assay may be started At the end of the Assay Wizard you also have the option of saving your configuration to an assay template file which is an excel worksheet containing the assay information Select the directory in which the template will be saved then enter a save name when prompted at the bottom of the scr
38. assigned to a cell type definition and not to a plate layout Also compounds and injection media are assigned on a port letter basis in addition to well location Database Functionality for Editing and Deleting Entries In the Assay Wizard the database feature is used to save the definitions of compounds and media used in the assay These definitions can then be used later by loading them from the database The figure below displays the selection dialog for browsing the database contents Edit and Delete buttons have been added to support database maintenance Existing entries can now be modified or completely removed Compound List Name COMPOUND 1 Stock Conc 250 00 uM COMPOUND 1 IWork Conc 250 00 nM COMPOUND 2 Database Dialog Where are the Definitions Stored When running this software for the first time it creates three files in the program directory The files are CellDatabase cif MediaDatabase mif and CompoundDatabase dif These files store the definitions of their respective content The software can be configured to access these files from a different location In addition these database files can be pre configured on a different computer and then copied to the instrument controller or vice versa To configure the software to access these files from a different location 1 Click on the Instrument button to enter instrument setup 145 Deahorse Bioscience 2 Click on the File Preferences but
39. based on the formulation of Dulbecco s Modified Eagle s Medium however no sodium bicarbonate buffering agent or glucose is present Media Specifications Volume 1L Osmolality 291 mOsm Kg H20 Endotoxin lt 0 0052 EU mL Storage 2 8 C Expiration This liquid media has data supporting a shelf life of 18 months when properly stored Media Constituents Media Component Final Concentration Mg as MgSOz 0 8 mM Ca as CaCl 1 8mM NaCl 143 mM KCI 5 4 mM NaH2PO4 0 91mM L Ala Gln Glutamax 2 mM Phenol Red 15 mg ml Preparation for use in XF Assay XF Assay Media contains no added glucose or sodium pyruvate To prepare media for an XF assay do the following 1 Warm media to 37 C in water bath 2 Add 100 mM Sodium Pyruvate Note this will be assay dependent 10 mL per 1 L of media 1 mM final concentration 3 Add glucose for the desired final concentration based on the following table 153 Deahorse Bioscience Final Glucose Grams of Glucose Concentration per1L 0 mM 0 00g 2 5 mM 0 459 5 0 mM 0 90g 10 mM 1 809 25 mM 4 509 Adjust pH of media to 7 4 using NaOH or HCI Filter sterilize if sterility was compromised Aliquot desired amount to use in XF assay The reminder should be stored at 4 C Re adjustment of pH may be necessary if stored for longer than 7 days Sie A Buffer Capacity Based on Glucose Concentration The buffer capacity of the XF Assay Media was determined for the following concentrations
40. clean sterilize solution bottle no check valve and red O ring should be placed in the left position facing the back of the PS Locate the bottle hose assembly and install using the pictures and diagrams as a guide The end with the red check valve is fitted onto the LOWER input media pump nozzle match green O rings Ensure the arrows on the valve indicate that the flow direction is out of the bottles and into the pump 25 Seahorse Bioscience Connect the 3 bottle connectors to the three bottles Use the color coding on the O rings and the diagram on the rear of the PS to facilitate accurate connections The insertion of the hoses gt into the pinch valves A which are the round k SA white valves on the rear Oo of the PS requires some STRETCH TORN force The hoses may be 2 SLIDE INTO HOUSING stretched to facilitate s 1 insertion into the pinch wy 2 valves see diagram to right The end of a wooden Q tip also may be used to facilitate insertion Fitting tubing into a pinch valve The tube must be stretched then slid into the pinch valve completely Note this is critical step If the hoses are not completely engaged in the valves the instrument will alow the flow of clean disinfectant solution or water when commanded to pump media Ensure that the media bottle contains the intake hose with the white check valve and be sure that the bottle intake hoses do not occlude the Blue vacuum filter con
41. compounds etc Seahorse Bioscience strongly encourages users to titrate the seeding density of cells as the initial optimization experiment Once an optimal seeding density has been obtained users may then move towards optimizing the concentrations of compounds to be injected media composition etc Using columns 1 6 on the cell plate for 6 different cell densities is a convenient and thorough method to assure optimal cell number 70 2 at ty Se Deahorse Bioscience 15 Fatty Acid Oxidation FAO Experiment using C2C12 myoblast cells Mitochondria oxidize a variety of fuels to generate ATP through oxidative phosphorylation Cells can utilize fatty acid glucose and amino acids as their fuels Traditionally fatty acid oxidation FAO assay is performed by quantifying radioactive byproducts H2O or COs after incubating cells with radio labeled fatty acids such as palmitate or oleate In this protocol increased oxygen consumption rate OCR immediately following exposing partially starved cells to palmitate substrate is used as an indicator for FAO in XF24 Analyzer This offers a rapid easy and non radioactive alternative assay for fatty acid oxidation In addition both kinetic and accumulative data can be obtained in a single assay Palmitate O 4 COs HeO 1 Reagents and Materials a Krebs Henseleit Buffer KHB see Appendix VI b Palmitate BSA conjugate and BSA vehicle control Appendix VI c XF24
42. fuse will disable the heater should it reach an abnormally high temperature The plenum also provides additional spill protection Should a spill occur outside the tray the fluid will fall through the air circulation grates and into the plenum A drain tube leads from the bottom of the plenum to the exterior of the instrument to prevent accumulation 30 b 5 eahors eb loscience 4 Basic Operation of the XF24 Instrument Power and Warm up To power on the touch screen controller press the power switch on the top of the controller To prevent accidental power down of _ the controller the switch may _ be disengaged in the Power Options menu of the Windows OS control panel under the Advanced tab Turn the instrument on using the power switch on the rear panel On the right side of the instrument near the access door there is an LCD message screen When the instrument is powered up the LCD message screen will show When the XF24 software is launched the LCD message screen will update and show the following display Note that it is recommended to allow at least 2 3 hours for the instrument to fully warm and equilibrate to the set temperature Measurements are taken using the XF24 Sensor Cartridge It has two analyte sensor spots on the bottom of each probe that are sensitive to oxygen and proton concentration respectively By measuring the concentration of each analyte over time the oxygen consumptio
43. gt 2 for 60 minutes to allow cells to pre equilibrate with the assay medium 7 Loading Sensor cartridge Proper orientation notch at lower left a Warm BSA and BSA Palmitate to 37 C prior to loading sensor cartridge b Load 75 ul of BSA control into 2 ports per well e g ports A amp C for columns 1 3 150 ul total per well c Load 75 ul of BSA Palmitate into 2 ports per well e g ports A amp C for columns 4 6 150 ul total per well Note optimal palmitate concentration for different cell tyoes can vary Palmitate concentration titration is recommended for a specific cell type line 72 Deahorse Bioscience 8 Protocol Commands Command __ Time min Port Calibrate J o _Equilibrate Mix LoopEnd _ Inject A inject C Loop Start 5X o Mx 3 o Wait 2 J Measure 3 o En Notes 1 To assay for the effect of FAO agonists such as metformin cells can be treated for the desired amount of time before performing the fatty acid oxidation assay Alternatively rapidly permeating compounds e g phenformin can be injected and rate measured before palmitate BSA injection 2 C2C12 myoblasts may be differentiated into myotubes by replacing 10 FBS in the growth media with 2 Horse serum and allowing growth for an additional 4 6 days Myotubes typically show higher OCRs and have a more robust response to added palmitate thus the Measurement time should be decreased to 1 5 minutes 9
44. in NADH from being converted to usable energy in the form of ATP In this case one will observe a decrease in OCR due to impaired mitochondrial function with a concomitant increase in ECAR as the cell shifts to a more glycolytic state in order to maintain its energy balance Note that Antimycin A can also be used here and is preferred by many researchers as it inhibits Complex Ill in the mitochondria and thus all O2 consumption is blocked 1 Reagents and Materials a Oligomycin FCCP Rotenone or Antimycin A Solutions Appendix VI b DMEM Running Media Appendix V1 c DMSO Sigma D8418 d Distilled Water Gibco 15230 170 e Calibration buffer Seahorse Bioscience 2 Growth Medium a 500 mL DMEM Gibco 11965 092 80 Deahorse Bioscience b 10 FBS Hyclone SH90070 03 c 5 mL Penn Strep Gibco 15140 122 d 5 mL Sodium Pyruvate Sigma S8636 e 5 mL Glutamax Gibco 35050 061 3 Seeding Protocol a Cells are seeded in XF24 cell cultures plates with 25 000 cells well in 100 ul of growth medium and placed in 37 C incubator with 10 COs b Cells will adhere to the XF24 cell culture plate within 1 hour c After cells adhere add 150 ul of growth medium and return to 37 C incubator with 10 COs d Assay cells in XF24 24 hours after seeding 4 Preparation of Assay Template Using the Assay Wizard Appendix I generate a template with the following group layout Columns 1 6 25 K C2C12 cells Group 1 CTL control
45. including Background Correction or blank wells need to have a control media or compound loaded in the port being used to ensure proper injection in all wells The notch on the bottom left of the cartridge denotes the front of the cartridge Note all compounds should be diluted with the appropriate running media DMEM KHB or RPMI before being loaded into the sensor cartridge The ports are loaded by angling the tips into the ports and gently loading making sure not to cause any leakage into the well below Do NOT insert the pipette tip completely into the port to avoid pushing the volume through the bottom of the injection reservoir Loading may be done with either a single channel pipette one port at a time or with an 8 or 12 channel pipette all ports in one column or row at once respectively 11 Calibrating the sensors and running the experiment Login to the XF24 software and allow the XF24 Analyzer to warm to 37C The XF24 Analyzer should be left on overnight so the instrument is at the proper temperature Set up an experimental template by using the Assay Wizard see Appendix l This template can be prepared in advance so it is ready to be loaded into the XF24 software Open the appropriate assay xls template by clicking the Open icon on the bottom of the screen and choosing the desired template If using the Assay Wizard to design an XF experiment just before running then this template will already be loaded When f
46. is enabled The examples in figure above displayed a single analyte Use the Y2 axis to display a second analyte on the graph as shown in the figures below comparing rates 4 and 5 OCR and ECAR vs GROUP Avg Rate 5 EELE and LUAR we HLHIP Awi GELI OCR pMoles min WAU HA aa OCR pMoles min ap ui Hdw yyvo3 IBEP Oa us CP 0 06_CCCP 0 125_CC S_CCCP 0 5 RUU GROUP Group Display o of OCR amp ECAR at Rate 4 before TET and Rate 5 EE injection 122 Deahorse Bioscience OCR and ECAR vs GROUP Avg Rate 5 A bar graph option is also available as shown in the figure to the right To enable 1 Click on the hw button to open the graph settings dialog ul Hdw yyo3 E 2 a oO o 2 Click on the Advanced Graph Options button 3 Enable the option to Use Bar Graph when GROUP is selected on X axis j 0 0 _CCCP 0 06_CCCP 0 125_CCCP 0 25_CCCP 0 5_CCCP 1 0_CCCP Note when in non bar graph mode the groups are displayed with connecting line making this a convenient way to look at trends However the X axis is scale is fixed by group only and does NOT represent a true linear scale i e it does not correspond to the listed concentration cell seeding or other increasing decreasing values Aligning the Y1 and Y2 origins for Base lined Data When displaying the base lined data for two analytes it may be useful to scale the Y1 and Y2 axes such that the origins are alig
47. modified assay configuration to an excel file using the Save Template button at the end of the wizard Load the assay configuration template into the software on the instrument controller before proceeding to step 2 Step d must be performed on the instrument controller 2 Select the Buffer Capacity calculation Technique The default value of Variable is recommended This step must be performed on the instrument controller prior to starting the assay If using the default Variable technique this step can be skipped since no change is required 109 Deahorse Bioscience Measuring the Experimental Buffer Capacity of XF Assay Media Buffer capacity BC will change as a function of the amount of buffering agent in the media For the XF assay media this includes the low concentration of phosphate as well as the presence of any added compounds that may accept or donate protons H including but not limited to glucose pyruvate glutamine Glutamax albumin or protein including serum Thus each media composition will have a unique buffer Capacity value expressed in mol L M For example the BC of XF DMEM with 25 mM glucose 1 mM pyruvate and 2 mM GlutaMax 1 Gibco 35050 061 8 52 x 10 M Note that BC will also change as a function of temperature of the media thus ensure that when calculating the BC you perform at 37 C The formula to determine the experimental BC is BC moles of OH or H added pH change volume o
48. of collection has not changed Applying the new algorithm recalculates the oxygen data taking into to account the Stern Volmer relationship and empirically generated time and rate constants for the behavior of Oz in polystyrene plastic 96 Deahorse Bioscience Data files captured with versions 1 3 of the XF software or earlier should be updated to the current Mult Assay Data Viewer XF 24 Files 1 F 95 Files Format To do this launch ene er the updater accessible Original Data iewer Format from the Seahorse Bioscience program group and select this Multi Assay format Then click on select Select Files and convert Select Directory and convert files and convert This will open a browser and cue allow file selection After file selection click OK and the file will be updated A new File Updater Utility file will be saved in the same location with a new suffix appended to the file name Note the original file will be kept and remain unchanged eahorseBioscience File Updater Software The File Updater utility can also facilitate upgrading a large group of files using the select Directory and convert command Files can also be updated using the Save As feature of the XF24 application software Data files captured with version 1 4 do not require updating To apply the new algorithm open the data file in Excel Under the Special Operations tab click on the Change O2 Calc Method button Swit
49. of glucose Media was prepared as described in Preparation for use in XF Assay Glucose Concentration Buffer Capacity 0 mM 0 0009092 5 0 mM 0 0008779 10 mM 0 0008862 25 mM 0 0008557 Note that Seahorse Bioscience suggests that serum e g FBS be omitted from the final media formulation as it will affect the buffer capacity of the media If FBS is required for cell viability or phenotypic maintenance Seahorse Bioscience suggests using low amounts 1 or less and determining the resulting buffer capacity empirically for accurate PPR calculations Please follow the instructions below should you wish to make your own DMEM XF Assay media A Reagents a Dulbecco s Modified Eagle s Medium Base 8 3g L Sigma D5030 1L b 100x 200mM GlutaMax 1 Gibco 35050 061 c 100mM Sodium Pyruvate Sigma S8636 Assay Dependent d Glucose Sigma G8270 Assay Dependent e NaCl Sigma 83014 f Phenol Red Sigma P 5530 B Dissolve DMEM Base in 500 mL dH20 Separately dissolve 1 85g NaCl in 500 mL dH20 Combine NaCl solution with DMEM Base solution Remove 20 mL from the combined solution Add 10 mL 100x GlutaMax 1 10mL 100 mM Sodium Pyruvate assay dependent and 15 mg Phenol Red Add glucose powder for desired 154 T sae aia GA i P Deahorse Bioscience concentration 4 5g 25mM glucose Warm media to 3 then pH to 7 4 using 5M NaOH Sigma Filter sterilize Media can be stored at 4 C for later usage KHB XF
50. protein content total DNA content or total mitochondrial number This could also be an ATP assay or cell viability assay Once the additional assay has been performed enter the normalization constants into either the XF reader or Excel software and click on the N button to view the normalized results For more detailed information on AUC and statistical analyses please see Appendix III Special Operations AUC Area Under the Curve and Statistical Analyses Functions For more detailed information on AUC and statistical analyses please see Appendix Ill Special Operations Graph Appearance The XF viewer allows the data to be displayed in several graphic formats For example one may view the kinetic data by plotting OCR and or ECAR PPR vs time Other displays include plotting OCR and ECAR PPR data by group see Appendix IV for more detailed information on the Group feature or plotting OCR vs ECAR PPR The advantages of using the group feature or OCR vs ECAR PPR graph is that total bioenergetic profile of the cells may be displayed on a single graph that is simple to view and interpret For example we begin with separate kinetic graphs of the OCR and ECAR PPR data HEUROHAL BEP 090708 HEURONALBEP090708 A2_782009_1416 NEURONAL BEP 090708 HEURONALBEP090708 A2_782009_1416 ocr Decar OCR pMoles min an 40 50 TIME min 40 50 60 TIME min OCR only ECAR only uiwu Hdw yvo3 E
51. the correct orientation as shown in the diagram above At the end of the pre programmed calibration procedure the user will be prompted to replace the Utility plate with the cell plate which should be incubating at 37 C without CO3 Remember to keep cell plate sensor cartridge and Utility plate at 37 C before running the assay The calibration will take approximately 30 minutes After calibration is complete continue to follow the instructions on the screen When prompted remove the calibration plate from the instrument when door is open and replace with the experimental cell plate again ensuring the plate is flat and in the correct orientation Note that the sensor cartridge remains in the instrument during this exchange Once calibration is complete and the assay has begun the Results button can be clicked to view the data while it is being collected When the run is complete the user will be prompted to remove the cell plate and sensor cartridge from the XF24 Analyzer All data will be automatically exported to the export file set up at the beginning of the assay 50 Aeahorse Bioscience XF Experimental Results Using the XF Data Viewer 12 Basic Operation of the XF Data Viewer and Graphic Analysis This section introduces the basic graphing data display and data analysis features of the XF24 Analyzer software both in the XFReader application and the Excel based Data Viewer Note that these programs fu
52. the mean squares of the two sources of variation If the null hypothesis is true F is likely to be close to 1 Larger F values correspond to smaller P values Again note that a significant P from the ANOVA test only indicates that one or more groups may be different from the rest It does not tell you which group or groups are different We use Tukey s HSD post test to determine which groups are significantly different from the others 121 Deahorse Bioscience Appendix IV Advanced Graphing Options and Features Selecting Group for the X axis The x axis drop down box has a GROUP option As shown in the right graph below when selected the group curve shows the difference in rates between the well groupings The x axis tick marks are labeled with each group name as specified in your assay configuration The groups can represent injected compounds treatment dosage cell type or any other factor OCR vs TIME Avg OCR vs GROUP Avg Rate 5 ocr 600 Goo moo m 0 1 G02 Gos E 1 0 OCR pMoles min pt OCR pMoles min 25 40 0 126_Ccc 6 CCCP oa pa in seas Changing X X axis from Time to TETA Example is CCCP titration uM with TET cells Note that the group curve is a snapshot of a single measurement of the assay Use the scroll bar in the lower left of the Results screen to scroll through the rates The rate number is shown above the graph Also this feature is only available when Well Group Mode
53. to insert defined steps steps are inserted only above the currently highlighted step blue bar Gaai Cole Medi RPP Compo Dmi anmi Cannesi Labea BETI Assay Wizard Protocol Screen Calibrate The first command for every XF assay calibrates the sensors for the experiment Equilibrate used immediately after calibration Default command consists of a 2 minute mix and a 2 minute wait repeated 3X This equilibration period allows the temperature of the culture plate and instrument to stabilize after the transfer of the culture plate to the instrument sensors used to oxygenate the cells and mix injected compounds e E Wait instructs the instrument to wait between steps Measure used for the measurement of OCR and ECAR Time of measurement is dependent on the basal OCR value of cells typically 3 5 minutes Inject instructs the instrument to inject compounds loaded in the ports into the wells The user may choose port A B or both Loop used to repeat a set of commands multiple times Used before and after the series to build a loop e g loop 4X mix 3 min wait 3 min measure 3 min will repeat the mix and measure commands 4 times Since most protocols consist of repeated cycles of Mix Wait and Measure commands protocol construction can be simplified by using a loop to repeat the same command cycle This will also shorten the length of the protocol command structure One drawback is that once a loop has st
54. used for simple and quick identification of the condition for each group and will be displayed in this manner in the results graph at the end of the experiment e g control vs compound Group Info Assigns group information to wells e g Compound name compound concentration Control vs test compound escalating dose series etc You can also customize the colors for each group There are 24 predefined colors To assign groups to wells a Click on Create New Group button b Enter a group name in the label space Assay Wizard Group Labels Screen t a X Dee Wee tne Wired temesnens s Assay Wizard Well Labels Screen c The color of any group may be changed by clicking in the colored box to the right of the Label space d Click the wells in the plate layout to add them to a specific group e Repeat to assign each well to a group if desired f Up to 24 groups can be identified for each plate Wells that are not assigned to a group will be black 90 Deahorse Bioscience Protocol Tab This is an especially important tab as it meer programs the instrument for the experiment with respect to timing of the E calibration mix measure and inject Pe rowcol sian commands Before entering any a commands the screen will look like the screen diagram to the right There are several command buttons that may be utilized to build a protocol To create a protocol use the buttons
55. 300uM palm E 150uM oleate 10000 00 E 300uM oleate EBSA 15000 00 pMoles min T i Q a O o gt e c lt o lt 5000 00 0 00 BSA 150uM 300uUM 150uM 300uM BSA palm palm oleate oleate TIME min AVG AUC SD AUC 5617 96 4693 26 13547 00 1746 24 22088 49 1961 85 11357 06 2387 78 16952 23 2179 89 3914 89 1548 18 fa ea 4 abt 6 8 9 10 11 15 16 18 19 28 29 150u 30 30 33 M 4 gt if Time Events Calibration AUC Anova OCR1 lt Ready The AUC worksheet contains several elements At the top left is a bar chart of the average AUC for each group The error bars indicate Standard Deviation or Standard Error depending on your selection in the graph settings dialog 118 horse Bioscience To the right is a copy of the Data Viewer graph Configure the graph before running the AUC Also make sure it is not scrolled off the edge of the Microsoft Excel application window You can also go back to the Data Viewer Worksheet and manually copy the graph to paste in this worksheet Directly below the chart is the table of data displayed in the chart Microsoft Excel XF24 Data x s BEA ial File Edit View Insert Format Tools Data Window Help Type a question for help 7 el Ee BREA IA ha TF Oe OL E 100 HEE ey Py EE iy is gA Ce Reply with Changes End Review 5617 96 4693 26 13547 00 1746 24 22088 49 1961 85 11357 06 2387 78
56. 4 into appropriate ports on the sensor cartridge Optimal injection volume is 75 ul All wells of each port used need to be loaded with either compound or media control including temperature correction wells If the sensor cartridge is allowed to cool down more than 5 minutes return to incubator for 10 minutes to allow it to heat back up before starting the calibration 7 Load sensor cartridge and Utility plate into the right side of the instrument tray 8 After calibration is complete replace Utility plate with the pre incubated cell plate Injection Port Layout and suggested Mix Wait and Measure times OCR Mix Time Wait Measure pmol min Time 25 50 2 min 2 min 5 min 50 100 2 min 2 min 4 min 100 500 3 2 3 500 1000 4 0 3 1000 Call Support Deahorse Bioscience Table of Contents Quick Reference XF24 Assay Protocol Table of Contents Introduction 1 2 Seahorse Bioscience XF24 Instrument Overview Technical Specifications XF Instrument Controller and PS Installation Procedure Unpacking and Component identification Suitable Locations for the instrument Removing the Shipping Cartridge Internal Components of the XF24 Set up and Interconnects Cable Installation Keyspan Adapter Installation and Configuration Unpacking the Prep Station Installing the PS Manifold and Connecting the Hose Systems Testing the Prep Station Temperature settings for the PS incubator Safety Considerations Basic Operation o
57. A 8 Protocol Commands Equilibrate Mix LoopEnd i Command _ Time min Port 3 Mix 3 En a E E E E a ee SS a ee _ T1 Aeahorse Bioscience 9 Analysis and Discussion As discussed above DNP and FCCP have predictable effects on the bioenergetic state of HepG2 cells The goal of this experiment is to determine the concentration of DNP or FCCP that will produce maximal OCR and ECAR response to these uncouplers Note that this method may be used for any acute acting compound to be tested Typical results for each compound are presented in OCR vs time and ECAR vs time graphs below For the DNP assay OCR vs TIME Avg pMoles min 0 0 5 0 10 0 15 0 z200 75 0 300 35 0 400 450 500 55 0 60 0 65 0 TIME min OCR vs Time For the FCCP assay OCR vs TIME Avg pMoles min f wT fi _ ole 10 00 20 00 30 00 40 00 50 00 60 00 TIME min OCR vs Time ECAR vs TIME Avg 50 410 0 15 0 270 0 25 0 3O0 34 0 400 450 S500 55 0 600 65 0 TIME min ECAR vs Time ECAR vs TIME Avg 10 00 20 00 30 00 40 00 50 00 60 00 70 00 TIME min ECAR vs Time 78 Deahorse Bioscience By plotting the response vs the log DNP or log FCCP in uM units one may obtain a typical dose response curve Dose Response for DNP with HepG2 cells Dose Response for FCCP with HepG2 cells 125 100 as c _ g 100 24 75 EC lt o 24 8 uM e S EC lt o 0 08 uM
58. Assay media A Reagents and Materials a NaCl Sigma S3014 dry or Sigma S6316 5M solution b KCI Sigma P4504 c MgSO Sigma M2643 d Na2HPO Sigma 83397 e Glucose Sigma G8270 dry or Sigma G8769 45 solution f Carnitine Sigma C0158 B Preparation of 5x KHB base from dry reagents To make 1 liter 5x KHB add the following reagents separately in the listed order to 900 ml dH20 in a 1 liter beaker containing a magnet stir bar Allow each to dissolve before adding the next one 5X mM __ 5X g L MgSO4 Na2HPO4 6 0 852 Adjust final volume to 1000 mL with dH2O then filter sterilize and store at 4 C for later use C Alternate preparation of 5x KHB base from stock solutions Use 5M NaCl stock solution Make 100 mL stock solutions of KCI 1 M MgSO 0 5 M and NasHPO 0 5 M in dH20 as follows NaCl 5 purchase aaa KCL J1 7 456 g MgSO 6 019 g NasHPO 7 098 g To make 1 liter 5x KHB to 833 5 mL dH20 add the following 155 5 eahorse Bios cience Stock concentration 5X Amount for 1 L M mM mL 111 mL 23 5 mL MgSO NaHPO 05 612m a es Filter sterilize and store at 4 C for later use Preparation of 50 mM carnitine stock Dissolve 81 mg carnitine in 10 mL dH20 in a 25 ml conical tube Filter sterilize and aliquot into 1 ml in 1 5 ml eppendorf tubes Store at 20 C Frozen stock is good for one month and may be used within three days of thawing when kep
59. Bottles Reagent Bottle Top Tube with white check valve Reagent Bottle Top Tube without check valve 15 Bottle Hose Assembly with red check valve Media Hose Assembly with red check valve Waste Hose Assembly with waste bottle top Vacuum Filter muffler Suitable Locations for the XF24 system The XF24 is designed for laboratory use The internal environment of the analyzer is controlled to a preset temperature by the user and therefore laboratory room temperature must be maintained within the range listed in the specification table Analyzer temperature control performance can be monitored using the status display on the right side of the analyzer The XF24 uses optical detection technology to measure extremely low levels of fluorescent emission from analyte sensors While the analyzer has been designed to 16 Aeahorse Bioscience shield room light excessive light such as direct sunlight is likely to influence measurements and should be avoided The XF24 system is intended to be used with a non COs 37 C incubator that is a built in component of the Prep Station This allows cell culture plates to remain warm during transport from the incubator to the analyzer Removing the Shipping Cartridge from the XF Analyzer The analyzer is shipped with protection components that must be removed prior to use To prevent damage during shipping the instrument is shipped with a cartridge loaded onto the probe
60. Cartridge currently has two Analyte Sensor Spots on the bottom of each probe that measure oxygen consumption and extracellular acidification Each well has four injection ports designated Port A Port B Port C and Port D The Cartridge Lot Number is on the front of the cartridge and the Bar Code is on the back The sensor cartridge is loaded into the instrument with the Bar Code facing the back lot number facing front XF24 Cartridge Top View with injection ports layout When preparing for compound injections its important to know the layout of the ports The ports are indexed as Port A B C D and are arranged as shown above Compressed air is used to force the compounds through the ports and into the wells Injections are performed on a port basis All A ports are injected at once as well as all B ports etc Also note that the entire volume from each port is injected Analyte Sensor spots Cartridge Lot Number Bar Code Cartridge Spring Injection Part XF24 Cartridge Bottom View 48 b 5 eahors eb loscience Load pre warmed 37 C injection compound into desired sensor cartridge port s prior to beginning calibration of the sensor cartridge The recommended injection volume is 75 ul but injections can range from 50 ul to 100 ul It is strongly recommended that each series of ports has the same volume e g all A ports are filled with the same volume all B ports are filled with the same volume etc All wells
61. Every other day after the initial change partial media changes are necessary 3 Aspirate media leaving 50 100 ul behind in each well Add 100 150 ul of fresh media to each well using the side of the well to avoid disturbing the monolayer Maintenance of Cells Growing in XF24 Culture Plates 1 Media can be fully changed up to 48 hours after initial seeding 2 After 48 hours a partial media change is suggested for monolayer integrity following the same recommendations detailed above Note that you may also use the Prep Station to prepare the cell plate for the XF assay Operation of the PS is described below 45 ahorse Bioscience Operation of the Prep Station for Media Exchange Turn on the Prep Station PS and allow the incubator and bottle holder to warm to 37 C It is recommended that the PS be turned on the day before the XF assay is to be performed or allow at least 2 3 hours for the unit to warm to 37 C Fill the PS bottles with the appropriate solutions see diagram to right Clean disinfectant solution dH2O and XF Assay Media f the assay media is cold 4C allow at least 2 hours for the media 500 ml to reach 37 A W SAL lt Launch the XF24 software log in and press the Prep Station Button in the lower right portion of the screen If the Clean and dH2O0 lines tubes are dry prime the lines with Clean solution and dH2 respectively Use the prime function sc
62. Ins Analysis ToolPak co The Add Ins dialog will appear Select the SS ped checkbox for Analysis ToolPak VBA Click Lookup Wizard OK C Solver Add in Analysis ToolPak Provides functions and interfaces for financial and scientific data analysis Using the Tool To use the AUC tool click on the Grid amp Granh Controls Data Files Special Operations AUC Gr oup T Analysis button i AUC Analysis Group Statisties AUC Anava Analysis Set Normalization count below the graph in the excel data file H Data Viewer Assay Configuration Level Dats Rate Data tate View Rate Data Tima Events Calibration Error Log lt 3 40 4 42 1 wa Bonk 113 Group AUC T Test Warning Only the wells selected in the grid are used for the analysis Current Assay NEURONAL BEP 090708 NEURONALBEP090708 A2_782009_ Select the analyte starting rate and end rate for me Renin och pie dete calculations Click OK after making your selections First Rate Base Line 1 End Rate Sheet Name A new worksheet will be added containing the results The dialog automatically provides a default name for the new AVG AUC SD AUC 1070 92 616 30 546 33 444 77 worksheet You can change the name 336 16 1 ta only by modifying the name of the newly 286 84 193 78 created sheet a N Md 4b H Rate Data Fi Time Events ri Calibration AUC ECAR1 i Analysis Results Th
63. Note that if the B baseline function is enabled in the Data Viewer the yellow region will have zero value assuming the function is using the third point as the baseline point Ei Microsoft Excel XF24 Data xls K ied File Edit View Insert Format Tools Data Window Help Type a question for help 5 Is bs BREA REFE MIA E a a A E 100 F G H J g 14175 09 14892 31 11573 59 24040 75 20111 SDev 0 00 0 00 T 0 00 f 0 00 430 38 1036 77 i 1171 67 i 790 83 936 47 J 2288 41 2941 89 j 1928 14 1139 08 2685 22 i 3931 27 i 2392 16 1189 65 2677 56 l 4402 03 T 2269 36 1080 63 l 2478 117 i 4701 51 2036 04 841 75 2380 92 J 4940 12 1940 52 P Value Anova P Value Tukey Post test 0 000005 0 021697 0 000024 0 135485 0 001171 0 020582 0 921403 0 668813 0 003492 0 270028 0 198877 Confidence level 1 P Value Anova Confidence level 1 P Value Tukey Post test 0 999995 0 978303 0 999976 0 664515 0 998829 0 979416 0 078597 0 331187 0 996508 0 729972 0 801123 72 M 4 gt mf Time Events Calibration AUC Anova ocR1 lt Ready nD nD DD On oan nonin 4 Saaz ass algieeigiaeiaieaieiasisisiaessesss ss The P value for the ANOVA test is presented in column A If the P is significant use the table of P values starting in column D to compare specific groups These P values are generated using Tukey s HSD post test Note that this tab
64. O Sigma D8418 d Distilled Water Gibco 15230 170 e Calibration buffer Seahorse Bioscience 2 Growth Medium a 500 ml MEM ATCC 31 2003 b 10 FBS Hyclone SH30070 03 c 100 U or ug ml Pen Strep Invitrogen 15140 122 3 Seeding Protocol a Cells are seeded in XF24 cell cultures plates with 30 000 cells well in 100 ul of growth medium and placed in 37 C incubator with 5 COs b Cells will adhere to the XF24 cell culture plate within 3 hours c After cells adhere add 150 ul of growth medium and return to 37 C incubator with 5 COs d Assay cells in XF24 24 hours after seeding 75 Aeahorse Bioscience 4 Preparation of Assay Template a Using the Assay Wizard Appendix generate a template with the following group layout b Assign all wells except A1 B4 C3 and D6 as HepG2 Assign Temp Corr to wells A1 B4 C3 and D6 c Below is the information for the stock and working concentrations of DNP or FCCP and group assignments Note that the concentrations are different for each compound pNP FCCP Stock Final in Assay Stock _ Final in Assay 0 03 DMSO 5 Compound Prep a Prepare a 1 0 M solution of DNP in 100 DMSO and a 3 mM solution of FCCP in 100 DMSO b Dilute solutions to make a 10x stock in DMEM Assay Medium for injections c For a final concentration of 300 uM DNP make a stock of 3 mM in DMEM Do a serial dilution of 1 part DNP to 2 parts DMEM 1 3 to get
65. Please see Appendix V page 140 for more details on setting up individual user preferences Set up an XF assay template by using the Assay Wizard Appendix of this manual details the steps to use the Assay Wizard to make a template This template can be prepared in advance either on the controller or on a separate laptop or desktop PC so it is ready to be loaded into the XF24 controller Note that the XF Software is MS Windows based and may be loaded and used any Windows compatible computer 33 Aeahorse Bioscience Introduction to the XF Software and Graphic User Interface The Run and Files screens look like this The XF24 Run Screen The XF24 Files Screen The user may open a previously constructed template by clicking the Open or Files icon and choosing the desired template Choose the proper folder on the left side of the screen and choose the file on the right side of the screen Alternatively use the Assay Wizard icon to launch the wizard which will take the user step by step through the assay creation process Navigating through the buttons and tabs across the top right 4 buttons 1 The Run button screen is for viewing the XF assay file that is currently open It has several tabs for information and user preferences a b Plate shows experimental set up for the cell plate Displays information well by well for cells well labels and media conditions Compound Displays injec
66. Point Rate Display error bars omitted for clarity seahorse Bioscience strongly encourages users to view their data in both middle and point to point modes and choose the format that best represents the data In summary if rates are stable relatively unchanged over the measurement time then using the middle point mode accurately represents the OCR If the rates change Significantly throughout measurement period then the data is most accurately represented using the point to point mode and further may provide enhanced biological information about the system used 105 a ite eahorse Bioscience Extracellular Acidification Rate ECAR Calculation and Settings Since the introduction of the XF24 extra cellular acidification rate ECAR has been calculated using the 2 Points technique for determining the slope The slope between the average of three points at the beginning and end of the measurement period determined the rate As shown in the figure below the calculation technique has been changed to Line Fit with an Edge Offset of 1 The Line Fit technique uses the same line fitting algorithm as the Fix Delta technique to calculate the slope However all the points in the measurement excluding those omitted by the Edge Offset are used GWR Seahorse XF 24 Current File Plate Compound Desc Protocol Info Do background correction Rates calculation settings uuc ECAR v Recommended Settings for Calculating ECAR
67. Similarly right clicking on a folder name or touching the folder icon will also display a popup menu Again close the popup by clicking or touching the Cancel button Double clicking with the mouse is also supported on either files or folders Double clicking on a file opens that file and displays the data in the Results section Double clicking on a folder opens the folder and displays the contents on the right hand tree display in the Files section 82007 _1421 xIs 817 0KT2006 _428 xIs 2006 _428 xls ee 2006 _545 xls 2006 _752 xIs J Phase III J Reference Assemblies CJ Seahorse Bioscience My Assay Templates _ My Data T FReader24 Delete waveGames p all Information ename TPublish ws Media Connect 2 Cancel 151 D Wi R eahorse Bioscience Option to Maintain Instrument Temperature when exiting XF Software In prior releases when exiting the software on the instrumen s touch screen controller the instrument heater WARNING WARNING was turned off allowing the system to cool In this release when clicking the Exit button the message prompt shown in Figure 3 will appear The user can ma choose to have the Instrument Maintain system temp while the software is closed temperature maintained while the software is closed or to turn off the heater Turn off heater System will cool until you start the software If opting to turn the heater o
68. Standard Deviation or Standard Error of the Mean rate plotting and various default graphing displays including graph title and graph setting X Y1 and Y2 axes This screen has a sub menu indicated by the Graph Appearance and Well Group Mode display options button clicking this button brings up a dialogue with more user defined preferences for the graph appearance including font size line width grid options and graph legend position 36 eahorse Bioscience File Preferences The Assay button allows user defined preferences for an assay template directory This is useful for opening templates and specifying a default template file and a default Protocol the protocol consists of the commands used to drive the instrument The Data button allows user defined preferences for exporting data files to a default location and associated file information including auto naming options for resulting data files This function is especially useful if the results are to be saved on a common computer server or if the instrument is available to the local area network The Database button allows the user to specify a location for cell type media type compound info and user databases These databases are accessible through the Assay Wizard CAP regres Per Baaksdes Biodeled Ia niar pee ort SMP rete Penta eee Braiena a ll Meade ey ED ba fag am a Ba par Bana bena LE adar H Rel al ry GETE TLA LEE de alee Pe ae
69. a stock concentration of 1 mM 333 uM 100 uM and 33 uM After injection the final concentrations will be 300 uM 100 uM 33 uM 10 uM and 3 3 uM respectively d For a final concentration of 1 uM FCCP make a stock of 10 uM FCCP in DMEM Do a serial dilution of 1 part FCCP to 2 parts DMEM 1 3 to a get a stock concentration of 10 uM 3 uM 1 uM 0 33 uM 0 1 uM After injection the final concentration will be 1 UM 0 33 uM 0 1 uM 0 03 uM and 0 01 uM respectively 76 5 eahorse B 1OSC1ENCE gt e Warm stocks to 37 C and adjust pH to 7 4 6 Cell Preparation a Remove growth medium from each well leaving 50 ul of media b Wash cells with 1000 ul of pre warmed DMEM running media remove 1000 ul as above and add 625 ul of DMEM running media 675 ul final c Incubate in 37 C incubator without COz for 60 minutes to allow cells to pre equilibrate with the assay medium 7 Loading Sensor Cartridge Proper orientation notch at lower left Warm compounds to 37 C prior to loading sensor cartridge Load the compounds into the injector ports as follows Column 1 Load 75 ul of DMEM 0 3 DMSO into ports A Column 2 Load 75 ul of 33 uM DNP 0 1 uM FCCP into ports A Column 3 Load 75 ul of 100 uM DNP 0 33 uM FCCP into ports A Column 4 Load 75 ul of 330 uM DNP 1 0 uM FCCP into ports A Column 5 Load 75 ul of 1000 uM 1 mM DNP 3 3 uM FCCP into ports A Column 6 Load 75 ul of 300UM 3 mM DNP 10 uM FCCP into ports
70. ab 85 Media and PPR Tab 87 Compounds Tab 88 Background Correction Tab 89 Groups and Labels Tab 90 Protocol Tab 91 About Mix Wait and Measure Times 93 46 48 49 51 99 67 71 19 80 85 Deahorse Bioscience End Tab and Saving XF Template Files 93 Quick Pick Text and XF Protocol Save Features 94 II OCR ECAR and PPR Information on Rates Calculations 95 OCR Calculations The Gain Fixed Method 95 OCR calculations The Level Direct AKOS Method 98 ECAR Calculations and Settings 105 PPR Calculations and Settings 106 Measuring the Experimental Buffer Capacity of XF Assay media 109 lil Group Statistics and AUC analysis 111 Group Statistics Feature 111 AUC Analysis using Multiple t tests 112 AUC analysis Excel Configuration 113 ANOVA AUC analysis 116 IV Advanced Graphing Options and Features 122 Topics Groups on the X axis Aligning the Y1 and Y2 axes Resizing Graph elements Displaying the assay date Custom graph titles Grid lines Graph Legends Injection Markers and Labels Time Axis Origin Options Interactive Adjustment of Axes Custom Text Labels Data Threshold Option 136 Working with Multiple Data Sets 136 Multi View and Multi Graph Display 139 Saving and Exporting Graph Data 141 Copying Saving and Exporting Graph and Well Grid Images 142 V The Media Cell amp Compound Database and 144 Additional Features and Options The media cell and compound databases 144 User Logi
71. al measurement cycle includes a mix wait and measurement period 4 What reagent is used for calibration 5 What is the proper temperature for all plates being run in the XF24 6 Sensor Cartridges need to be hydrated for before being run in the XF24 A 30 minutes B Overnight C 1 hour 7 True or False Compounds are loaded into the sensor cartridge before calibration 8 What is typically the shortest measurement period 9 True or False A 2 Step seeding procedure is used for seeding cells in the XF24 cell culture plates 10 When seeding a new cell line in XF24 cell culture plates how long should you wait before adding the final amount of growth media 11 What are the minimum and maximum volumes for each well when starting an XF24 assay 12 How many times can an XF24 sensor cartridge be run on its matching cell plate 13 What is the recommended volume of compound to load in each port 14 After which number measurement is the baseline typically stable 15 True or False The Bar Code on the XF24 sensor cartridge should be facing towards the user 16 True or False The seeding surface of the XF24 cell culture plate is the same size as a typical 96 well plate 163 5 eahorse Bios cience 17 After removing the growth media from a cell plate and replacing it with unbuffered running media where should the plate be stored 18 What is the Seahorse Bioscience Customer Support email
72. apacity will be treated as a fluctuating factor in the assay or a constant The following is a guide to configuring the settings that affect the PPR calculation To avoid redundancy references are made to other sections of this document for specific instructions 1 Specify the buffer capacity of the assay s running media This can be done several ways a With the Database feature introduced in Appendix V page 135 the software comes with four common pre defined running media When opting to load a running media from the database the listing will include DMEM 25 mM glucose DMEM 11 mM glucose MDC RPMI and KHB These definitions have pre set buffer capacity values Follow the steps for Recalling a Running Media Definition from the Database on page 138 Appendix V b Alternatively follow the steps for creating a new assay media definition on page 83 Appendix c To specify multiple buffer capacities in different wells or groups of wells on the plate assign multiple media to the plate from the Media Layout section of the Media amp PPR tab d Lastly instead of using running media in the assay configuration specify a value in the Default Buffer Capacity field in the Calculation Settings section If no media has been designated as described in the above steps this default value will be used instead for all wells Steps a b or c can be performed on a computer other than the instrument controller If doing so save the
73. arted you will not be able to edit the contents of the loop Media Change This button is not yet functional in the current version of software 91 eahorse Bioscience Copy Copies the highlighted commands can be used in conjunction with the Paste command to facilitate protocol set up Unselect unselects the highlighted command Paste Pastes the copied commands into the protocol ABOVE the command highlighted in blue S Modify allows the user to modify the highlighted command with respect to time or port injected Delete Removes the selected command s from the protocol p f Clear All Removes ALL commands from the protocol Load Loads an entire saved protocol into the screen Useful if a number of protocols will be used for various assays A Click on the desired command on the right to enter it into the protocol on the left of the screen When the command on the right is selected a pop up screen will appear to enter the time of each command or which injection port is to be used etc B Every protocol must start with the Calibrate command Seahorse Bioscience recommends then performing an Equilibrate command to allow the cell plate to thermally equilibrate after being loaded into the instrument C After calibrate and equilibrate commands are Protocol Start lI 4 Calibrate Probes i 2 Equilibrate 9 3 Loop 4 times HL 4 1 2 3 4 Mix for 3 Min 0 sec 5 Time Delay of 2 Min
74. ary to assign the injection media However if the injectate is prepared in a media other than the assay running media it may be assigned here Background Correction Tab This screen is used for choosing and m defining the Background Correction pE parameters for an XF assay template Sarre Do Background Correction this ack oe oe pets button engages the background correction function and should always be ON Label for background wells Typically Bkg Cor is used but the label may be changed by the user if desired a Paes fea La bal l t Assay Wizard Back Corr Tab 89 Aeahorse Bioscience Form a group with background correction wells When engaged default this instructs the software to automatically form a group using the Background Correction wells chosen Note that even if a group is not automatically formed the data and calculations will be performed properly Group ID User may choose any group number Label for Background group defines a label for the Background Correction group Groups and Labels Tab Used to assign groupings of cells compounds conditions etc Well Labels Assign specific info to wells e g cell type seeding number WT or KO etc Enter the desired label and highlight the wells to the right Click Assign to Wells This label will appear under each well Note this does not have to be the same as the info provided in cells media or compounds This can be
75. as a framework with which to build more complex experiments aimed at understanding various compound effects on cell metabolism mitochondrial function and overall bioenergetic aspects Typical results of this experiment are presented below for both OCR and ECAR 82 090122 Bioenergetic_Profile MF PK_1222009 952 xIs _ Ecar 100 00 30 00 30 00 70 00 60 00 50 00 OCR pMoles min ECAR mpH min 40 00 30 00 20 00 OCR vs Time ECAR vs Time Here we observed the expected responses in OCR and ECAR as the cells are treated with each successive compound For oligomycin OCR decreases as a result of blocking ATP synthesis at mitochondrial Complex V Since the cells are unable to synthesize ATP via OXPHOS they revert to glycolysis to meet their demand for ATP thus we observe an increase in ECAR As shown previously FCCP acts as an uncoupling agent Since the cells must now overcome the proton leak across the inner mitochondrial membrane OCR increases significantly as more Oz is consumed to pump the excess protons across the mitochondrial membrane Finally rotenone inhibits mitochondrial Complex I which causes the flow of electrons to cease in the electron transport chain and thus the consumption of Oz is drastically reduced Beyond the expected changes in respiration and ECAR a number of respiratory parameters may be obtained from this data This is summarized in the figure below Here we see that we may OCR v
76. asurement cycle is performed for 2 5 minutes During this time the media is gently mixed and is followed by a short temperature recovery period The analyte levels are then measured until the oxygen concentration drops approximately 20 30 and media pH declines approximately 0 1 0 2 pH units The measurement is performed using 24 optical fluorescent biosensors embedded in a sterile disposable cartridge that is placed into the Seahorse 24 well tissue culture microplate Baseline metabolic rates are typically measured 3 4 times and are reported in pmol min for OCR and PPR and in mpH min for ECAR Compound is then added to the media and mixed for 5 minutes and then the post treatment OCR and ECAR measurements are made and repeated As cells shift metabolic pathways the relationship between OCR and ECAR PPR changes Because XF measurements are non destructive cells can be profiled over a period of minutes hours or days The XF24 system include a bench top analyzer and touch screen controller disposable measurement compound delivery cartridges and microplates for cell culture calibration media and XF24 software A plate preparation station PS for exchanging media in the tissue culture plate may be purchased separately Consumable cartridges microplates and calibration reagents are packaged and sold in sets named Fluxpaks One Fluxpak contains the materials for 18 assays z ae aia A za P F eahorse Bios cience 2
77. ation curve tends to be quite sharp and too much FCCP can actually diminish responses in OCR Typical ranges to test would be 0 1 1 0 ug ml of Oligomycin 0 1 5 0 uM FCCP 0 1 1 0 uM Rotenone Note that the responses to each compound above especially FCCP will be influenced by the assay media composition base type glucose pyruvate presence absence of BSA etc Further if the XF assay media composition is changed optimization will need to be re performed The presence and concentration of pyruvate is especially important in obtaining the maximal respiratory capacity due to FCCP Seahorse Bioscience has observed in a number of cells lines that omission of pyruvate abrogates the ability of cells to respond maximally above baseline to FCCP Typically concentrations of 1 10 mM pyruvate should be tested to understand the optimal concentration of pyruvate to obtain maximal respiration Note that pyruvate AND glucose may need to be cross titrated to obtain the optimal media conditions for the experiment For more information and details about this experimental method and the idea of spare respiratory capacity please see Bioenergetic Analysis of Cerebrocortical nerve terminals on a microgram scale spare respiratory capacity and stochastic mitochondrial failure by S Choi A Gerencser and D Nicholls Journal of Neurochemistry 2009 109 pages 1179 1191 84 Aeahorse Bioscience Appendices Appendix
78. ay Rate Type selection OCR First Rate Base Line 7 End Rate 10 Alpha to use for ANOVA 0 05 Available groups Groups for ANOVA BKG COR OF R 25 0 OFR 25 1 OFR 25 2 CTL 25 0 CTL251 lt lt Remove lt lt CTL252 Sheet Name PAUCAnvaOCRT S OK Cancel 117 eahorse Bioscience A new worksheet will be added To the workbook containing the results A default name for the new worksheet Is assigned You A2 78 AUC Anova OCR Rate T 10 AKOS ols 1 2 1 3 DURDONA BEP OPO TER IEURCERAL BLP ODE can modify the name of the J AVG AUC Measurements 7 10 5 Goca 4 s00 00 aT a newly created sheet E mm za tooo 300 wens f Serre NW 3 em ans ora 2 Bertin 13 m 5000 00 E o i 6 www c 1 Gi a je www Bo al ig r 20 5 t p Lees q 4 8 wom 0 00 eee 22 000 00 a t t ph at 1 4 5 S TIME gees xs ium 10072 la 1144 5p 68 P 3 129 23 538 11 Sro 3 79 59 3128 1 x 1011 2 33 Ea k D Rate y 000 a a Wf Level Gata J Rate Gata Gite ew Z Rate Guta Tene Gverts Calteation Z iko Reay Analysis Results Microsoft Excel XF24 Data xls Cc ex ied File Edit View Insert Format Tools Data Window Help Type a question for help bo Ee oe 1 ie j A aR E OER EN i 10 i F ERER Z 1 AUC Anova OCR Rates 3 9 2 AVG AUC Measurements 3 9 30000 00 25000 00 EBSA 20000 00 E 150uM palm E
79. ay conditions to wells if applicable A gi g q a m aA O Add to injection ports of Load Ports cartndge and Calibrate Transfer plate w4 l Cartridge to XF24 Prepare stock compounds if needed freshly made Aeahorse Bioscience Quick Reference XF 24 Analyzer Protocol Please refer to the XF24 Manual for detailed instructions 1 Pre incubate sensor cartridges overnight in XF24 Calibrant solution in a non CQOs incubator at 37 C 2 Leave XF24 Analyzer on overnight with XF24 software running and logged in to ensure temperature is at 37 C on the day of the assay 3 Warm all media and compounds to 37 C and adjust pH to 7 4 Filter to ensure sterility 4 Perform XF Assay media change on cell plate and incubate in a non COsz incubator at 37 C for 60 minutes before running on XF24 Analyzer When performing media change leave behind approximately 50 ul of media in each well and rinse with 1 ml XF Assay medium Final volume per well should be 500 1000 ul 5 Load XF Assay template into XF24 software or make a new template using the Assay Wizard Use the table below to program the optimal mix wait and measure cycle times When working with a new or unknown cell line start with a 2 min mix 2 min wait and 3 min measure cycle Verify instrument protocol commands export preferences and any instrument settings that may have changed 6 Load 50 100 ul pre warmed compounds diluted in XF Assay media at pH 7
80. can be recalled later and added to an existing assay configuration The Assay Wizard screens for defining growth media cell type running media compound and injection media all support this feature in the same way To demonstrate the instructions below will refer to running media but they apply similarly to the other assay materials that are part of the assay configuration Storing a Running Media Definition to the Database 1 In the Media Info screen of the Media amp PPR tab click on the Add Media button 2 Select New Media in the popup screen 3 New Media Info will appear highlighted in the Running Media List on the left On the right click on and edit the fields to define the media 4 Click on the Add to Database button to store the definition Recalling a Running Media Definition from the Database 1 In the Media Info screen of the Media amp PPR tab click on the Add Media button 2 Select Load from Database in the popup screen 3 A window with a running media list will appear Click on and highlight the desired media name from the list The definition will be displayed on the right side window Click Select 4 The selected media should now appear in the Running Media List on the Media Info screen 144 Deahorse Bioscience 5 To complete the configuration go to the Media Layout screen and assign the media to the appropriate locations of the wellplate Note Unlike running media a growth media is
81. ch the method from Gain Fixed to Level Direct AKOS Note that you may take data captured in the 1 7 software and revert to the Gain Fixed algorithm as well as modifying edge offset and delta parameters by first switching to the Gain Fixed method in the Excel data file You may then load this file into the XF Reader software and access the Gain Fixed parameters in the Settings tab see figure on page 93 above Grid amp Graph Controls Data Files Special Operations AUC Analysis Group Statistics AUC Anova Analysis Set Normalization count Change 02 Calc Method 97 Aeahorse Bioscience Data Viewer Special Operations The default OCR setting in the 1 7 XF24 software is the Level Direct AKOS algorithm The settings tab looks like the figure to the right Note that there are no settings for edge offset and delta as these parameters are not applicable for this algorithm Also while the volume indicates 7 ul a value of 22 7 ul is used by the algorithm to reflect the diffusion and storage characteristics of the well plate Please see Akos Gerencser et al Analytical Chemistry 2009 for details about how the 22 ul volume was empirically determined and the rational for using this value in the algorithm The Gain Fixed Method Do background correction Rates calculation settings EMA TEGH 7 8e 004 Volume Technique Cont Avg v Level Direct AKOS setting
82. dance with the goal of the assay and the user s preferences One common way to troubleshoot unexpected results is to look at the level data Level data is the raw data corresponding to the changes in mm Oz vs time or pH vs time for OCR and ECAR respectively Perform the following actions 1 Set the graphing options for X axis time Y axis Oo or pH 2 Pick a single well to analyze e g C2 3 Turn OFF the averaging function You should now see a graph that looks similar to this Oe Mime This graph represents the change in 02 ee oxygen tension mm Hg of Os with we A B C D respect to time in a single well Ideally one would expect to see little variation in the starting point of each measurement see yellow dashed line indicating that the O2 sensors have an adequate amount of time during mixing and waiting to re equilibrate to ambient Os concentration in the media Inspection of the Y axis scale indicates 20 ie ee d that variation in the starting O2 tension 0 0 10 0 20 0 30 0 40 0 50 0 60 0 70 0 among measurements iS minor about 5 mm Os which is within the error specifications of the instrument Note that the starting Os level is approximately 158 mm Hg which is the accurate value of Os partial pressure Also note that injections do not affect the Os levels 140 0 120 0 100 0 02 mmHg x a 64 r i ra ra L iie fd ri a tat rt Far 0 0 E 0 0 10 0 20 0 30 0 40
83. e Deahorse Bioscience Air filter replacement At approximately one year intervals the air filter at the rear of the analyzer should be replaced The filter retainer snaps away from the housing use a screwdriver or wooden stick to remove it ii Drip tray replacement Drip trays snap to the floor of the moving plate carrier Lift the try upward and remove from the right side of the analyzer 4 A eahorse Bioscience Performing the XF Assay 5 Seahorse Bioscience Cell Culture Overview In order to obtain accurate and reproducible data from XF experiments it is imperative that good cell culture techniques are established and practiced in a consistent and reliable manner The techniques for thawing passaging and seeding cells washing and changing media and making reagents have been employed and optimized for use with the XF instrument system Most of these techniques do not differ significantly from what most cell culturists practice however we have found that following these protocols in a strict manner allows for tight control of experimental variables associated with cell biology studies and assists with troubleshooting when problems do arise Most important adherence to these protocols usually ensures success of the XF experiment In other words the better one practices upstream cell culture the more reliable reproducible and accurate the downstream XF data Please refer to the following sections to familiari
84. e rate If you modify the rate calculation parameters the level data graphs will update to reflect any changes in the points used In the top figure the Oz level data is shown for two measurements When calculating OCR there is an edge offset of 1 so the first point of level data is omitted and is displayed in the graph as an outline TIME min Level Data The next four points are solid They fall within the 50 mmHg drop in O2 level that is the range used for the OCR calculation OCR vs TIME Avg The last four points being beyond the 50 mmHg range are also Outlined and omitted from the calculation In the figure to the right the rates corresponding to the level graphs are displayed G L r g 25 Note this configuration of level data TIME min only applies to level data captured using the 1 4 version of XF Reader Corresponding Rates software that uses the Gain Fixed algorithm This configuration does NOT apply to data captured with the 1 7 software using the Level Direct AKOS algorithm see below Benefits of this Setting By discarding the first data point more linear reproducible measurements with less bias between columns are achieved High rates introduce non linearity to the data set This is normally found in the first two data points during which time the gradient across the 100 eahorse Bioscience sensor is changing The degree of nonlinearity is wor
85. e AUC worksheet contains several elements At the top left is a bar chart of the average AUC for each group To the right is a copy of the a Sr Gee in Bo we wa mY we We Data Viewer graph h a ce ation eatin i ie a Cae Te oi j C 114 i Aeahorse Bioscience Below the chart are tables of the area information on an individual well and rate basis Each section is color coded by group color And below that is a table of P values followed by summaries of the t test calculations for each combination of groups la TISECEETECSTESEnEEELECA 5 i 4 7 S2 ba 22772 vit is Calculations To calculate the area under the curve each rate is multiplied by its time interval to determine the area under that part of the curve The area for the rate chosen as the baseline for the AUC calculation is then subtracted from the area of each subsequent rate The results are then summed to provide the total baseline subtracted area of the curve lf working with the absolute rate data the area will be in units of pmols for OCR and mpH for ECAR However if the Data Viewer is configured to display the data in percent baseline mode then the AUC is calculated using the percent values and not the raw pmol min or mpH min rate data In this case the area would be in units of and would represent a total percentage change from the baseline In addition if the normalization option is enabled and the data is
86. e Prep button in the lower right of the screen The PS Operations screen will appear Click on the Prime Manifold Tab Click on the CLEAN button and press the green Start button Check the liquid color and flow in the tubing to ensure that the manifold is being primed from the correct bottle Repeat the test priming for the H20 and Media bottles Next perform a mock media change to test the system Click on the Media Change button Engage the Do Prime and Do Rinse buttons Load an XF24 culture plate onto the PS Press the green start button and set the final volume per well to 675 ul The PS PS Change Media Screen will prime the manifold from Media 21 Deahorse Bioscience bottle then perform 1 aspiration and dispense cycle There will be a pause in the operation then the PS will perform a second aspiration and dispense cycle Do not remove the cell plate until all 2 cycles are complete After the 2 wash dispense cycle is complete the PS will automatically enter into a self cleaning mode that consists of washing the manifold with H2O clean disinfectant solution and H20 again e Once proper assembly has been confirmed rinse the bottles with water e Install the spray shield and tighten the thumb screws To install the spray shield ensure that the metal tabs on the right side of the shield are aligned with the holes in the middle of the PS where the spray shield inserted Slide the incubator bac
87. e done via the User Manager tool in the Instrument setup section 146 Seahorse Bioscience Seahorse Guest New User User Login Screen Adding a New User From the login screen create a new user by clicking on the New User button In the User Info dialog enter in the login and password Though only three seahorse icons are available there is no limitation on the total number of users that can be created Current User Info Login Seahorse User 1 Password Confirm Password User Icon Startup preferences Cancel User Login Info 147 Deahorse Bioscience Click on the Startup preferences n Run Screen v button to display this dialog The Start Screen setting determines the initial screen the user sees after logging in C Program Files Seahorse Bioscience XFReader24 Options include the Run Screen Files Assay Wizard Run Start and Prep Default Template File B a The Assay Template Directory and the Default Template File can also be specified These are the same settings as found in the Assay section of File Preferences Assay Template Directory Editing Users Display Preferences File Preferences After several user logins have been added it may be necessary to remove or edit Administration them This administrative function can be done from the User Manager section of Plate Manager Instrument setup Display this section by selecting the User Manager but
88. e well grid would show values from the first data set Grid amp Graph Controls Data Files Special Operations File Name 0713 07 16 11 49 FAQ F24 ASSAY DATA 1x 0713 07 16 1639 ted assay data 2 sls Clear List The Data Files tab showing use of checkboxes and highlighted listings Averaging Multiple Data Sets The Average Profiles button on the Grid amp Graph Controls tab allows you to average data from multiple data sets The averaging feature requires that the two data sets be from similarly run assays with the same number of measurements and identically named groups The figure below shows a group from two different data sets graphed separately on the left and averaged together on the right OCR vs TIME Avg of Selected Profiles OCR vs TIME Avg OOCR OOCR 1000 A amp C A amp C E 800 800 E 3 E 600 S 600 z 400 65 400 O L O 200 200 0 20 40 60 80 100 0 20 40 60 80 100 TIME min TIME min Averaging Data from Two Data Sets 137 Deahorse Bioscience Multi View and Multi Graph Display Overview To provide more flexibility in viewing data using the Data Viewer in Excel up to four different views of the Data Viewer can be configured Any combination of display options can be applied to each view such as different data sets different graphing options or with different selected wells Toggle between the view using tabs located at the upper right Initially each data file will start out w
89. ed Save Graph Data This features takes the data currently displayed in the graph and exports it to a new excel worksheet By using the controls on the Data Viewer the graph becomes a data filter and this feature provides an easy way to export that data for additional analysis When done from the Data Viewer in Excel it adds a new worksheet to the existing data file However this can also be done from the application software The user will be prompted to enter in a filename and a new excel file will be created with the generated worksheet As shown in the figure below the worksheet will contain pictures of the well grid and graph The data will be located below the pictures organized by group or well depending on how the graph is configured 140 Microsoft Excel XF24 ASSAY DATA 1 xls L fox z P 1 7S Eg ile Ed vien Insert nat Tools Data Window Help Type a question for help zo O X TP Ly PF SW hi 75 oy n 4 Se E E E carmen meee emcees ___ __ _ _ ____s__1_4 __ F FAO XF24 ASSAY DATA 1 xIs OOCR 1000 OCR pMoles min g TIME min OCR 7 pMolesimin m Assay Indez TIME min Std Dev Std Dev Std Dev Std Dev EE EN EA EH EH EN EN EN 0 Em ER EH 14 EA EN EH EA 20 ER Ea 23 24 25 i 28 29 30 EB 32 33 34 35 36 38 39 40 TH 42 KA E M 4 gt wf Rate Data Plate View Graph Data1 a Ready Export from the Save Graph Data function
90. ed separately as ptc files for later recall This feature is only available in the Assay Wizard Appendix II OCR ECAR and PPR Information on Rate Calculations With the release of the XF reader software version 1 7 a new algorithm has been implemented for calculating oxygen levels and consumption rates Users will have a choice of using the original algorithm Gain Fixed or the new algorithm Level Direct AKOS The Direct method is now the default standard Each will be described below Oxygen Consumption Rate OCR Calculation methods and Settings OCR Calculations The Level Direct AKOS method Overview XF software release 1 7 includes an improved algorithm for calculating oxygen consumption that increases the useful range of OCR the temporal resolution and durations of measurements The original algorithm employs a constant value for the volume of oxygen temporarily trapped in the well chamber during a measurement In reality the environment is more dynamic with oxygen able to enter the well chamber through various boundaries As shown by the red arrows in the figure to the right oxygen can pass through the sides and bottom of the well plate as well as through and around the sensor sleeve Oxygen Diffusion in the Well 95 A eaghorse Bioscience As the cells consume oxygen from the Probe measurement chamber a series of gradients are formed that cause oxygen to flow across these bo
91. edia to each well then pipetting to remove again leaving behind approximately 50 100 pl of media Add desired volume of XF assay media to each well to achieve the desired Starting volume typically 500 1000 ul Incubate the XF24 cell culture plate in a 37C incubator without CO until ready for use or at least 60 minutes prior to loading plate in XF24 instrument to allow cells to equilibrate to the new media Note You may also remove media and media wash using an aspirator hooked to a 5 ml pipette however you must take care to leave approximately the same amount of media in each well 50 100 ul It is important NOT to remove the media completely to prevent exposure of the cells to air and potentially drying out The key is to be consistent with the amount of media left in the wells 44 Deahorse Bioscience Place a 5 ml aspirator tip into the corner of the well far enough to reach the top of the media line but without touching the bottom of the well Aspirate media leaving 100 ul behind in each well See picture below Hold tip of pipet here allowing aspirator to remove media After changing the growth media to XF assay media load the appropriate XF template file onto the instrument software so it is ready to start just after loading the sensor cartridge with compounds Media Changes for Differentiation in XF24 Cell Culture Plates 1 After 24 hours perform a full media change into differentiation media 2
92. een The saved template can be modified at any time by opening it back up in the Assay Wizard and changing the desired fields To end the Assay Wizard press the End Wizard button Improved Text Input in the Assay Wizard Quick pick Text When using the Assay Wizard text entry can be repetitive and tedious The same text is often used in multiple assays To make it easier to fill in text fields entries will now be logged and then made available in future sessions for selection via a quick pick feature in the virtual keyboard Pisa ss th ta ts appa ta Use the quick pick feature by AEE oOo v clicking on the down arrow button found to the right of the entry box on the virtual keyboard red square If the button is disabled then there are no prior recorded entries available to select from You will only be able to select from prior text entered into that specific field So it will take time to build up a catalog of entries for each field The Virtual Keyboard Logged text will be saved to the file TextDatabase tdb This file will be stored in the location specified by Info Database in the Database section of File Preferences found under Instrument setup If this file is deleted all recorded entries will be lost and the file will be started again Protocol Save Feature 94 When constructing protocols in the Assay Wizard Load and Save buttons will be available to load and save protocol files Protocols can be sav
93. ells This experiment demonstrates changes in overall cellular bioenergetics using three different compounds that perturb cellular respiration The purposes of these protocols are several fold 1 Introduces new users of the XF24 to the experimental methods used to obtain cellular metabolic measurements including proper cell culture techniques optimal experimental design preparation of reagents and solutions gathering data using the XF24 instrument interpreting data using XF Reader software 2 Provides a set of reference or control experiments to access experimental user technique biological systems instrument performance and software performance These experiments can thus be used for troubleshooting 3 Offers a starting point of methods to study and investigate cellular bioenergetics including mitochondrial function metabolic pathways identifying mechanisms of action drug effects and toxicity etc 66 gt Deahorse Bioscience 14 Cell Titration and Uncoupling Experiment using C2C12 myoblast cells In this experiment C2C12 myoblast cells are seeded at three different densities in Seahorse cell culture plates The basal oxygen consumption OCR and extracellular acidification ECAR rates are measured to establish baseline rate The cells are then metabolically perturbed by addition of FCCP 300 nM final FCCP Carbonyl cyanide p trifluoromethoxyphenylhydrazone is an ionophore that is a mob
94. es will be listed DMEM 25 mM glucose ae Z DMEM 11 mM glucose KHB and RPMI Note that these media have their respective buffer capacity values defined see below for more info on buffer capacity Media layout Assigns running media to individual wells Enter the information the same way as assigning cell information to wells described above Be sure to specify the volume of running media used Typically this will be 500 1000 ul and be dependent on the number and volume of compound injections 2 Ta 2 Went tae Wuse beet mee et Assay Wizard Media Layout Screen PPR Proton Production Rate PPR is an alternative measurement of Extracellular Acidification Rate ECAR It is reported in pmol min same as OCR and requires that the buffer capacity of the media be defined and entered into the software for accurate calculations When defining the running media in the assay configuration media info tab a field is available for specifying the buffer capacity of the media at pH 7 4 This value is then used in calculating Proton Production Rate PPR Note that if no Buffer Capacity value is assigned to a new assay media then a default value will be used for the PPR calculation 87 Aeahorse Bioscience The Calculation Settings section is to support additional options for calculating PPR Note that if a Buffer Ba e Capacity value is defined in the Media M a ws screen this value will be used for calculat
95. f buffer tested so BC NaOH L ApH L Example You wish to determine the BC of your XF Assay media Materials and Reagents e Accurately calibrated pH meter e 1 0 M NaOH solution e Aliquot of XF Assay media warmed to 37 C e g 25 mL Volume of media tested 25ml 25x10 L NaOH added 27 ul 27x10 L A pH 1 18 from 7 40 to 8 58 recall pH is a unit less value Buffer Capacity 1 0 M 27x 10 L 1 18 25 x 10 L 27 x 10 6 moles 29 5 x 10 L 9 152 x 10 moles L 9 152 x 10 M 110 Deahorse Bioscience Appendix Ill Group Statistics and AUC analysis Statistics CVs and Data quality Because the XF24 Is a non invasive instrument multiple measurements may be made within a well and thus remove biological variability From measurement to measurement within a well the CV is 5 and can be considered instrument noise only Further because basal metabolic measurements are taken before addition of any compounds each well may serve as its own control Variability between wells that includes instrument plus biology for the average trained user is lt 20 This is composed of lt 10 for well to well variation and overall lt 20 from plate to plate or day to day Thus the majority of CV is inherent in the cells and operator By careful experimental design pipetting technique and proper cell culture and seeding methods this error can be minimized to give extremely consistent and accurate data Group S
96. f the XF24 Instrument Power and Warm up XF Software and Login Introduction to the XF software and Graphic User Interface Cleaning and routine maintenance Performing the XF Assay 5 6 Seahorse Bioscience Cell Culture Overview XF assay Day 1 Seeding Cells in XF24 Cell Culture Plates XF assay Day 1 Preparing Sensor Cartridge for the XF24 Assay XF assay Day 2 Preparing Cell Plate for the XF24 Assay Media Changes and Maintenance of cells growing in XF plates 45 10 12 30 42 42 42 44 44 Deahorse Bioscience 9 Operation of the Prep Station for Media exchange 10 XF assay Day 2 Loading the Sensor Cartridge with compounds 11 XF assay Day 2 Calibrating the sensors and running the experiment XF Experimental Results Using the XF Data Viewer 12 Basic Operation of the XF Data Viewer and Graphic Analysis 51 Well Grid Features and Options 53 Basic Graphing Features and Options 55 13 Analysis of XF Experimental Data General Guidelines Basic Analysis 59 Intermediate Analysis 60 Advanced Analysis Level Data 62 Sample Protocols for the XF24 analyzer 66 14 Cell titration and uncoupling experiment C2C12 myoblast cells 15 Fatty Acid Oxidation Experiment using C2C12 myoblast cells 16 Dose Response Experiment using HepG2 cells 17 Bioenergetic Profile Experiment using C2C12 myoblast cells Appendices I The XF Assay Wizard and Creating XF Assay Templates 85 General Tab 85 Cells T
97. ff restarting the software will turn the heater back on lf the heater is left on the instrument firmware will continue to safely regulate the system temperature Safeguards are in place to prevent overheating Cancel the Exit lf the Exit button is pressed by mistake click on the Cancel button Improved Data Recovery when Auto save Fails At the end of an assay if the software is unable to save the data to the specified target folder the software will instead save the data files to the Recovered Data folder in the software installation directory Figure 31 shows the error message that will be displayed if this occurs lf the software has been installed to the default location the Recovered Data folder can be found here C Program Files Seahorse Bioscience XFReader24 Recovered Data X Error 1060 The specified data folder was not accessible for the save Look for the result file in the Recovered Data folder 152 at aia A ra P eahorse Bi OSCLENICE Appendix VI Reagent and Solution Preparation XF Assay Media Specially Formulated DMEM 0 mM Glucose Part No 100965 XF Assay Media is for use in conjunction with the Seahorse Bioscience XF Analyzer instruments XF Biosensor cartridges and XF tissue culture plates XF assays require a non buffered media to accurately measure proton production rate and extra cellular acidification rate PPR and ECAR of cells growing in culture The constituents are
98. g cartridge Horne alter engegrig cartrige Lower cartridge in calibrant and wat lt i Mix calibrart for o Wat afier mixing for 2 Do calibration check for min Parere Wash cartricige for Subsystems Calibration Subsystems Background Correction 38 Aeahorse Bioscience Background Correction Clicking the button will show a dialogue that allows the user to assign wells for temperature correction Highlight the wells and click OK The current recommendation for Background Correction wells is A1 B4 C3 and D6 This information will already be set if the Assay Wizard Template is set up correctly Temperature Control A submenu for control over the temperature of the instrument Currently this may be set from 33 C 37 C This information is also displayed on the LCD screen on the side of the analyzer The instrument is typically set to 37 C must be engaged for proper temperature control Analyte settings Allows user to gather information of both O2 and pH or just one analyte Looking at only one analyte will allow the instrument to gather more data points per unit time Sounds You may download your favorite sound a wav file to notify you when the instrument has completed a step and needs attention from the user e g plate change from calibration to cell plate Ral Background Conoco Do background correct jn Subsystems Analyte Settings 39 Aeahorse Bioscience
99. g the XF software click on Refresh to expose the drive letter in the Files screen to allow immediate access to the device The File Menu Bar oat Paste x A R Eh SaveAs P 4 I Navigate to the parent folder of the current folder with the Up button Create new folders with the new button Copy and paste individual files to different folders or copy and paste entire folders and their contents to new locations Update file or folder names using the Rename button and remove unneeded files or folders with the Delete button Save your data The Save button replaces the Export button in previous versions Use Save As to save to a new filename File Filter screen you have a few options for _ iene anen e are deped A Aco files can be displayed or just those files generated by the XF software The data files come in two formats xls and xfd At the bottom left of the Files py Data amp Templates xls All XF Data amp Templates xls y 150 eahorse Bioscience Pop up Menus Temperature and Auto Data Recovery Popup Menu and Mouse Support When interacting with the list of files you can right click on any filename using your mouse to display the pop menu for quick access to some of the standard menu bar functions Click on the Cancel button to close the popup Using the touch screen you can also touch the file icon to the left of the filename to display the Popup
100. graph as shown in the figure to the right To add tt 1 Click on the button to open the graph settings dialog and then on Advanced Graph Options 2 Edit the Graph Title field Displaying Grid Lines Graph with a customized Title Grid lines can be displayed on the graphs in several different configurations To display them 1 Click on the button to open the graph settings dialog and then on Advanced Options 2 Configure the Grid settings Options include single axis lines or lines in both axes The lines can be either solid or dotted The figures below show four examples with grid line options Graph Examples showing Grid Lines in only one axis 125 Graph Examples showing Solid and Dotted Grid Lines Displaying a Graph Legend A legend can be displayed on the graph as shown in figure below To display the legend 1 Click on the button to open the graph settings dialog and then on Advanced Graph Options 2 Enable the Display Legend when in Well Group Mode options Also select the default location for the legend 3 Once displayed adjust the position of the legend by clicking and dragging 126 Deahorse Bioscience Graph Legend Examples Displaying Injection Markers amp Custom Label Options for Injection Markers The vertical injection markers can be hidden on the graphs as shown on the right in the figure below To hide them 1 Click on the bw button to open the
101. graph settings dialog and then on Advanced Options 2 Disable the Display Vertical Markers option Graph Examples with the Injection Markers displayed and hidden The injection markers in the graphs are labeled with the port letters corresponding to the source of the injection The port letters can now be replaced with customized labels The figure below shows a graph with the standard port labels and with custom labels FAO XF24 ASSAY DATA 1 xIs FAO XF24 ASSAY DATA 1 xIs OOCR OOCR iia DOSE1 amp DOSE2 3 E x ox O QO s O 0 20 40 60 80 100 TIME min TIME min 127 Aeahorse Bioscience Standard and Custom Injection Marker Labels Modifying the Labels To customize the labels e Se v dunu DOSE1 1 Click on the iw button to open the graph settings dialog and then on Advanced Graph Options 2 Click on the Injection Markers Options button to show the dialog seen in the figure to the right 3 Select the Label option Port letters or labels can be displayed or both 4 Edit the fields with the custom labels Fields will only be displayed Edit Injection Marker Labels for those ports used in the assay Options for Time Axis Origin The origin of the time axis defaults to the first recorded tick of data Options are now available to set the origin to some other point in the assay such as a particular protocol command or measurement To change the origin 1
102. head and lowered onto a plate on the tray These items must be removed prior to running your first assay by doing the following 1 Power on both the controller and instrument Launch the XF24 Analysis software The software will initialize the instrument and raise the probe head 2 Click on the Instrument button and then the plate manager button as shown to the right 3 Click on the strip cartridge button The tray on the instrument will open and you will be prompted to put a plate in the tray Since there is already one present just click OK 4 The shipping cartridge will be stripped from the probe head and then the cartridge and plate will be ejected from the instrument When the tray opens you will be prompted to remove the cartridge and plate Once removed click OK 17 horse Bioscience Internal components of the XF24 Analyzer The measurement enclosure is the heart of the XF24 Analyzer Removing the front cover reveals the measurement chamber in which the assay is conducted The electro optics hardware is enclosed in a card cage in the rear chamber and this is connected to the probe head through a set of bundles of fiber optic cables The base of the enclosure contains the primary controller board and heater assembly The following describes some of the hardware features pointed out in the image below 1 LCD Displays current instrument action and internal temperature 2 Cover Relea
103. hod has been implemented for determining HSD Honest Significant Different to generate a table of P values comparing each pair of groups Using the Tool 1 Click on the AUC Group Anova Analysis button 116 horse Bioscience 2 In the dialog select the analyte and starting and ending rates for the analysis Note You must also select which groups to include in the ANOVA analysis It s important to exclude unrelated groups such as the Temp Corr wells 3 Highlight groups in the left column and click Add to move them to the right The alpha variable is the significance threshold for P It defaults to the standard value of 0 05 Also one way ANOVA assumes groups are categorized based on one factor If there are multiple factors among the groups such as treatment and cell type or treatment and seeding number it is not valid to include both types of groups in the same analysis 4 Once the groups have been selected click OK Group AUC Anova Analysis Warning Only the wells selected in the grid are used for the analysis Current Assay Tn ae RE ee Rate Type selection OCR First Rate Base Line 7 End Rate 10 Alpha to use for ANOVA 0 05 Available groups Groups for ANOVA gt gt Add gt gt lt Remove lt Sheet Name FaucanvaOCRT o OK Cancel Group AUC Anova Analysis Warning Only the wells selected in the grid are used for the analysis Current Ass
104. ience XF24 Cell Culture Plate Dimensions Plate geometry 24 Wells 18 mm pitch Length L 127 8 mm 5 031 inches Width W 85 5 mm 3 366 inches Height H 20 3 mm 0 799 inches CTR L 18 9 mm 0 744 inches CTR W 15 7 mm 0 618 inches P f f i Plate material Polystyrene Cell culture compatible Proprietary Tissue Culture treatment Sterility Radiation treated Well volume 1 mL or less 500 1000 uL typical working volume Measurement volume V7 7 uL 11 horse Bioscience 3 XF24 Instrument Controller and PS Installation Procedure Unpacking and Component Identification The XF24 system is packaged in two boxes three boxes if a Prep Station unit is included On receipt immediately check each box for damage Shipping damage must be reported to the transportation company and Seahorse Bioscience using the contact information on page 2 of this document The following items are included in an XF24 system XF24 Analyzer The analyzer is a temperature controlled instrument that contains all optical and electronic measurement components to measure oxygen and proton flux of cells grown in XF24 cell culture plates The analyzer is used in conjunction with XF24 sensor cartridges Controller Operation of the Analyzer is done through a high resolution color LCD touch screen that may be located in front or beside the XF24 analyzer a separate keyboard and mo
105. ience Baseline Button Baselines the data to a user defined point typically the measurement just before compound injection and presents the data and graph in a format For more complex Baseline methods the point used for baseline reference can be changed Base Subtraction Methods Assay NEURONAL BEP 090708 tt t iti C S SCs YS and or multiple points may be used for Pot Poit2 Pot3 Poitd Baseline subtraction To access more eu o bo Po po Baseline options click on the Graph options a E dialogue button A dialogue will appear that will contain more settings for Baseline Baseline Subtraction Methods Excel options Normalization Button Allows users to normalize rate data after the assay has been performed Data may be normalized to cell count protein or DNA content cell viability etc This is especially useful when working with primary and or differentiated cells where the user may have difficulty determining an accurate cell number before the XF assay Normalization data can be entered interactively into the data file This can be done using the Set Normalization Count button in the Special Operations tab The Set Normalization Count button a RE brings up the entry dialog shown in the Select the wells you want to set the normalization cell court diagram to the right If multiple data sets are loaded you can select the data set from the drop down box at the top Click on
106. ile ion carrier It is an uncoupling agent because it disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase Complex V This leads to a rapid consumption of energy and oxygen without the generation of ATP In this case both OCR and ECAR will increase OCR due to uncoupling and ECAR as the cells attempt to maintain their energy balance by using glycolysis to generate ATP 1 Reagents and Materials a FCCP Sigma C2920 b DMEM XF Assay Media see Appendix VI c DMSO Sigma D8418 d Distilled Water Gibco 15230 170 e Calibration buffer Seahorse Bioscience 2 Growth Medium a 500 mL DMEM Gibco 11965 092 b 10 FBS Hyclone SH90070 03 c 5 mL Penn Strep Gibco 15140 122 d 5 mL Sodium Pyruvate Sigma S8636 e 5 mL Glutamax Gibco 35050 061 3 Seeding Protocol a Cells are seeded in XF24 cell cultures plates at 12 500 25 000 and 50 000 cells well using columns 1 2 3 4 and 5 6 respectively in 100 ul of growth medium and placed in a 37 C incubator with 10 COs b Cells will adhere to the XF24 cell culture plate within 1 hour c After cells adhere add 150 ul of growth medium and return to 37 C incubator with 10 COs d Assay cells in XF24 instrument 24 hours after seeding 4 Preparation of Assay Template Using the Assay Wizard Appendix generate a template with the following group layout 67 A eahorse Bioscience Co
107. ing concentration WC E a E is the final concentration of the compound Assay Wizard Compound Info Screen 88 Aeahorse Bioscience after injection The stock concentration SC is the concentration of the compound PRIOR to injection Injection Media This is the description of the media that the compound is diluted in typically this would be the same as the running media Add compound injection media by clicking the Add Media button and filling in the information on the right Repeat for multiple injection media Note that it is very important to pH the injection solutions to match as closely as possible to the pH of the running media to ensure accurate ECAR values Compound Layout Assigns compounds to individual wells Highlight desired compound on the left enter the compound volume highlight the Injection Media for Iese aiis that compound Choose port A B C or D Brorenone io OOOO above the plate layout then highlight which L e E wells will be receiving that compound gt Eh E E E e Click Assign to Wells Repeat for multiple compounds If multiple ports are being used for compound injection each port 7 needs to be assigned separately Be sure to specify the volume of the compound to a om be injected Assay Wizard Compound Layo n FF I ut Screen Available injection media Typically the injection media matches the running media e g XF assay media and thus it not necess
108. inished with loading the compounds into the injection ports if applicable press the large green START button and you will see a screen similar to that shown below Verify the current protocol If necessary change any commands using the icons located on the bottom left of the screen To change any of the information located on the right side of the screen touch the box you wish to change and a keyboard will appear to type any additional information Note to make sure the Save Directory and Save Name fields contain the appropriate information Experiment Ready to Start Screen 49 horse Bioscience Once the assay has started it is still possible to change commands that have not yet been executed To do this simply modify delete or add commands as desired using the action icons Ensure that the Cartridge Utility plate is FLAT on the tray and sitting between i e not on all 8 of the plastic plate alignment tabs see red arrows indicating 2 of 8 tabs NOTE that the notch in the cartridge should be located to the front and left side lower left corner in the photo above and the bar code should face towards the rear of the instrument Proper positioning of the XF Cartridge and Utility Plate on the Instrument Tray Click the Start button and follow the prompts on the screen for sensor cartridge calibration When the loading door opens place the sensor cartridge and Utility plate on the tray flat and in
109. ions even though the default value will appear in the calculations tab Please see Appendix Il for buffer Capacity determination and rate calculation settings Setting the PPR Media and PPR Calculations Tab 1 In the Media Info screen of the Media amp PPR tab click on and highlight an existing media name in the list If no names are listed click on the Add Media button to add one to the assay configuration 2 Click on the Buffer Capacity field located on the right side of the screen and enter in a value 3 Ifa new Running Media definition was created go to the Media Layout screen Click on and highlight media name in the Available Media list Select the wells that will be using the media Enter the volume and set the volume units Then click on the Assign to Wells button Multiple media with differing buffer Capacities can be assigned to the plate The XF software will default to using the Variable technique as shown in Appendix Il This is the recommended option and is especially important if using highly glycolytic cell lines Compounds Tab Compound Info provides a working list of compounds substrates agonists antagonists drugs etc that you may use for your experiments You may specify the stock and working concentrations here To add a compound to the list click New then enter the compound Name and concentration information Repeat for multiple compounds ROTERONE 10000 nM Typically the work
110. ith one tab representing the default view More tabs can be added using the buttons at the lower right This feature is useful for looking at several different graphs from the same assay at one time Microsoft Excel XF24 ASSAY DATA 1 xls Bl JEJ 81 File Edit View Insert Format Tools Data Window Help Type a question for help f X Se MEFE BO FS E OEA RU Arial 10 Bo 0 EnA Se ce A E ERS jy pe Reply with Changes End Review B A45 v ie et ee ae ae a a E a a ea E a ee E Dual Grid Graph A l 500 0 580 co 380 478 G 399 790 477 cS 145 404 g 522 AEG 452 398 42 36 if 62 14 7 2 GM3 Gm4 rp Grp 6 Grp 9 Grp 10 EA Grp 12 Grid amp Graph Controls Data Files Special Operations Grp 7 Grp amp OCR ECAR PPR a vy Eo DA D OCR ECAR H Well Group Mode Ea Average Profiles SJE gt i i e ww w t9 RS MIN b ab ab ab ad ab ab ad ad j w Nh N wN N wN olo N on e wn A v x Data Viewer Assay Configuration Level Data Rate Data Plate View Rate Data Calibration ja gt Ready NUM Data Viewer 138 Deahorse Bioscience Adding Multiple Views To add more views click on the d button Up to four views can be added By default the views will be numerically labeled 1 to 4 To remove a view select the a view tab and click on the E ropriate button View Tabs with Default
111. k to its normal position e Ensure that the temperature controllers both reach 37 C e When an experiment is to be performed fill the bottles with the appropriate solutions e Important Note All fluid lines need to be primed for the unit to dispense properly e g If water line is empty media will flow back in the wrong direction To prime the lines enter the Prime Manifold screen see above and select the bottle reagent to prime in the following order o A Clean o B HO o C Media 28 Deahorse Bioscience Temperature Settings for the PS Incubator The media and incubator temperature controllers are factory set to 37 C and calibrated for this temperature Changing the temperature set point does not guarantee that the temperature of the media or incubator is at the set point and it is recommended that a thermometer be put into the incubator to obtain an accurate reading if the temperature of the incubator is changed Green OFF PV lt SP ON PV SP Flashing PV gt SP Yellow OFF Normal Operation ON Control Setup Flashing Instrument Configuration Red Flashes when alarm s active Scroll Up Down Note that the displayed temperatures are the temperatures measured by a sensor on the outside of the media bottle holder and a sensor in the incubator It does not necessarily reflect the temperature of the plates in the incubator or the media in the media bottle Note that the typical warm up time for 500 m
112. l of medium from 4 C to 35 C is approximately 90 minutes compared to 2 hours in regular water bath Changing the Temperature Settings see diagram previous page e Open the incubator door and press the scroll key twice until SP is displayed e n several seconds the current set point will show on the display e Use the up and down keys to change the setting Note that the range of available set points is from ambient to 50 C e Press the scroll key twice until PROC is displayed The controller will enter normal operation in several seconds where the temperature of the controller feedback sensor is displayed 29 Aeahorse Bioscience Safety Considerations Safe operation of the instrument requires that the cover be securely attached and plate tray door is closed This also prevents heat loss and system cooling which can affect data quality The door opens automatically when the tray is extended allowing the operator to insert or remove the assay consumables When the cover is securely attached and tray door is closed a physical switch is engaged to monitor the system An optical sensor is used to determine the status of the door The XF24 has a heater that maintains a stable internal system temperature Typically the temperature will be maintained at 37 C as monitored by temperature sensors and controllers embedded in the tray and above the tray Two fans circulate air through the plenum containing the heater A thermal
113. le should be disregarded if the P value from the ANOVA test is not significant To the right of the table of P values is a table of Q values These values are from the Studentized Range distribution Tukey s HSD test is based on this distribution a oa t my Pee ete y diban AO Awe Ore 120 Microsoft Excel XF24 Data xls Secs 7a File Edit View Insert Format Tools Data Window Help Type a question for help X Be a oh 7 Oh Anova Single Factor 80 81 83 30 84 85 86 SUMMARY Variance 22471 83368 5617 95842 22026734 91 40640 98514 13546 99505 3049351 723 91 300uM palm 66265 46801 2208848934 3848842772 150uM oleate 34071 17491 11357 0583 5701508 495 300uM oleate 50856 6922 16952 23073 4751928 525 15659 56341 3914 890853 2396875 78 SS MS F P value F crit 806689759 3 161337951 9 20 91919663 4 83018E 06 2 958248913 107974095 1 77T12435 364 914663854 4 Notes on AUC Calculations 106 The area under the curve is calculated as follows For each rate the group s average is multiplied by the time interval for that rate This represents 1NGithe araa undar that nart of the cuna The araa fnr M 4 gt Time Events re Calibration AUC Anova OCR1 lt Ready At the bottom is the output from the Microsoft Excel ANOVA function Displayed are the P value and F value as well as the critical F value required to meet the specified alpha The F value is the ratio of
114. lumns 1 2 12 5 K cells Columns 3 4 25 K cells Columns 5 6 50K cells Group 1 12 5 K FCCP Group 2 12 5 K control Group 3 25 K FCCP Group 4 25 K control Group 5 50K FCCP Group 6 50K control Column and Group assignments Well Grid Layout 5 Compound Prep a Prepare a 1 0 mM solution of FCCP in DMSO Appendix VI b Using Running Media dilute a 1 0 mM FCCP solution to 3 uM to make a 10X stock solution for injection 0 3 uM final mix thoroughly c Adjustment of pH of the 3 0 uM FCCP to 7 4 is usually not necessary as FCCP does not appreciably change the pH at these low concentrations 6 Cell Preparation and Media Change a Remove growth medium from each well leaving 50 ul of media b Wash cells with 1000 ul of pre warmed DMEM running media remove 1000 ul as above and add 625 ul of DMEM running media 675 ul final c Incubate in 37 C incubator without CO for 60 minutes to allow cells to pre equilibrate with the assay medium 7 Loading Sensor Cartridge Proper orientation notch at lower left a Warm FCCP and media controls to 37 C prior to loading sensor cartridge b Load 75 ul of FCCP into port A for rows A and B c Load 75 ul DMEM running media DMSO 0 3 into ports A for rows C and D 68 Aeahorse Bioscience 8 Protocol Commands Command _ Time min Port Calibrate J o ECCE r Loop Start 3X J m o Wat 2 J Measure 3 LoopEnd J Inject A Loop Sta
115. ly to Vwaste bottle 24 Seahorse Bioscience Identify the waste hose assembly and locate the Y shaped waste manifold hoses portion Connect the two waste manifold hose connectors to the two Se i if i a Twist the 2 hose ends 1 2 turn to Media Hose amp Waste hose center ot t MANKO the left to facilitate hose assembly connected to They screw in with a attachment manifold half turn Connect the single end of the waste hose assembly to the vacuum pump connector on the rear of the instrument match black O rings Attachment of media hose to Identify the media hose the output of the media pump y Attachment of the media hose ep nor zie whietOnnde assembly Attach the k A ea cre Note waste hose connected to end with the twist fitting vacuum pump connector left to the connector on the side end of the manifold match black O rings Attach the other end of the media hose assembly with red check valve to the UPPER output nozzle of the media pump match white O rings Ensure the arrows on the red check valve indicate that the flow direction is out of the pump and into the manifold Next unpack and install the three bottles and caps into the bottle holders The media bottle with white check valve under cap and brown O ring should be placed in the right bottle position facing the back of the PS The H2O bottle and cap no check valve and blue O ring should be placed in the center position The
116. mage of the graph Save As 1 Right click on the graph and Save in E RES select Save Graph from the D My Documents av My Computer obup menu 9 My Computer HA My Documents Peper amp J My Network Places fy Shortcut to My Assay Template 9 Desktop Icons 2 The Save As dialog will Misc appear as shown in the maia figure to the right Enter in a gt filename and choose a file File name paean 99 T N Save type Save as type TIFF File tif kd Cancel iw Use enhance mode for the save Image will be 34 bigger 3 Select Use enhanced mode for the save for a larger image size i Create Image without border save As dialog for the Graph 4 Select Image without border to have a borderless image This produces a result similar to Copy Standard when choosing to copy and paste the graph 143 2 at aia S za P F eahorse Bi OSCLENCE Appendix V The Media Cell amp Compound Database and Additional Features and Options Option to Save Media Cell amp Compound Definitions Many users take advantage of the Assay Configuration worksheet template to define and store information regarding the cell tyoes media and compounds used in assays However the process of creating these definitions and copying them from template to template can be time consuming A Database feature has been added to the Assay Wizard to allow the separate storage of these definitions Once stored they
117. mber of protons can be calculated As the number of protons increase over a measurement period the PPR can be calculated The Fixed technique assumes the pH changes are small and that a fixed buffer capacity value is sufficiently accurate for calculating PPR over the range of pH values seen in the assay In this case ECAR will be directly proportional to PPR since no variation in buffer capacity is assumed The software defaults to using the Variable technique as shown in the next figure This is the recommended option and is especially important if using highly glycolytic cell lines General Cells Compounds Temp Correction Groups amp Labels Protocol End Media Info Media Layout Default Buffer Capacity mol L for Media 7 80e 004 Buffer Capacity calculation Technique Variable v PPR Calculation Settings in the Media amp PPR tab Configuring PPR The Media amp PPR tab of the Assay Wizard contains the settings for configuring how PPR will be calculated To calculate PPR the buffer capacity for the running media used in the assay must be specified This can be done using the Media Info and Media Layout sections as discussed earlier in this document The value specified in the Default Buffer Capacity field is used if no running media has been defined or assigned 108 at ty Se Deahorse Bioscience to the plate The Buffer Capacity calculation Technique determines whether buffer c
118. mizing the XF system for any new cell type that you will use in XF assays Typically one would optimize the following parameters cell seeding density response to metabolic compounds e g Oligomycin FCCP 2 DG rotenone etc mix wait and measure cycle timing and the concentration of injected compounds B Basic Analysis To simplify the initial analysis begin with the following settings e Select all wells and ensure that all the group boxes assigned are highlighted Check Enable Well Groups Engage Group Average Function Disengage Baseline and Normalization Functions Set graph to plot OCR vs Time or ECAR PPR vs Time In the Graph Options Dialogue select the preferred statistical parameter SD or SEM Set Rate Data Display As Middle Point Only Check Link Y1 of graph to display variables Check Do Temperature Correction Check Show Graph Legend Check Draw Vertical Markers LN NNN AN 60 Deahorse Bioscience Now looking at the OCR and ECAR data begin your analysis as follows How do the basal OCR and ECAR PPR data appear Do the results indicate a steady basal or baseline rates before injection Does the OCR and ECAR PPR data make sense i e what are the absolute rate values do the cells respond injected compounds as predicted and are there any obvious trends or changes in the rates Panning through the rate data what is the magnitude of the error SD or SEM A
119. n and User Profile Preferences 146 Refresh File Menu and File Filter 150 Pop up Menus Temperature and Auto Data Recovery 151 Aeaghorse Bioscience VI VII VIII Reagent and Solution Preparation XF Assay medias DMEM KHB 153 Stock Compounds oligomycin FCCP DNP Rotenone 2 DG 157 Preparation of Palmitate BSA complex 157 XF Cell Plate Coating Methods XF24 Trouble Shooting and FAQs XF24 Training Class Test Support Contact and Ordering Information 153 161 161 162 164 Deahorse Bioscience Introduction 1 Seahorse Bioscience XF24 Instrument Overview and Intended Use The Seahorse Bioscience XF24 instrument measures the rate of change of analytes currently dissolved oxygen and pH in the media immediately surrounding living cells cultured in a microplate Changes in the extracellular media are caused by the consumption or production of analytes by the cells Therefore a sensitive measurement of the media flux can be used to determine rates of cellular metabolism with great precision and in a totally non invasive label free manner A unique feature of the XF technology is its ability to make accurate and repeatable measurements in as little as five minutes This is accomplished by isolating an extremely small volume less than 10 ul of media above the cell monolayer Cellular metabolism causes rapid easily measured changes to the microenvironment in this small volume Typically a me
120. n rate OCR and extracellular acidification rate ECAR may be calculated Please see Sections 7 and 8 below for details on cell plate and cartridge preparation 31 eahorse Bioscience XF Software and Login Launch the XF24 software and allow the XF24 Analyzer to warm to 37 C for at least two hours Ideally the instrument should be left on overnight to ensure consistent and accurate temperature Note that the instrument will NOT warm to 37 C unless the XF24 software is launched simply turning on the instrument only will NOT engage the heater The following screen is displayed upon launching the software 8 XF_Software Standard Seahorse Apps XF Cell Stress Test Kit v Start App The XF24 Start Up Screen Click on the Standard button to go into the Standard software package and the User Preference screen If you are running a kit choose the Seahorse App of choice and click on the Start App button Refer to specific kit user manual for more details 32 Deahorse Bioscience Current User Info Login Password Confirm Password User Icon The XF24 User Preference and Dialogue Clicking on the Seahorse Guest button will take the user directly to the XF24 Run screen Clicking on the New User Button will open a dialogue to allow users to choose a preferred start screen assay template directory and default assay template It also allows for password protected templates files and data
121. ncludes extra data viewing analysis and statistical features These features are presented in detail in Appendix Ill 59 at aia A LA Deahorse Bioscience 13 Analysis of XF Experimental Data General Guidelines Now that you are familiar with the XF analysis tools and have produced an experimental data set the next question becomes How do analyze and interpret my XF data Obviously data analysis will be dependent upon the design and ultimate goal of the XF experiment however a few general guidelines and a simple methodology are provided to facilitate the analysis process A Before Analysis Begin with a well designed experiment practice good tissue and cell culture techniques and optimize the XF assay for the particular cell type you are investigating a Obtaining a consistent monolayer but not necessarily confluent of cells is vital to obtaining consistent and accurate data Using the recommended 2 step seeding method Section 6 will facilitate this process Look for even spacing of cells with no large cell clumps or blank patch A well designed experiment typically begins with a specific question or goal to the investigation Further a well designed experiment contains the appropriate controls that will facilitate and guide downstream analysis Optimization upstream will greatly enhance the ability to obtain meaningful and reproducible results downstream Seahorse Bioscience always recommends opti
122. nction in a very similar manner and either can be used for data display and data analysis The Excel version supports additional functionality e g statistics multiple file viewing and averaging other additional data display and information features Grid E Graph Contis Da i Hire Sect Opericce rd j OCA ECAR E OCRECAR Results screen in XF Reader Data Viewer screen in Excel The first part of this section briefly discusses the Results screen tabs in the XF Reader then focuses on basic graphing and analysis tools in the Excel format More detailed information on advanced graphing and data analysis tools may be found in the Appendices 51 a ite A s gt gt eahorse Bioscience Results Screen Tabs in the XF Reader Grid Graph Protocol Settings On the Results screen in the XF Reader screen there are tabs in the upper left Dual Grid Graph Protocol and Settings The Protocol and Setting tabs are identical to the corresponding tabs on the Run screen During an assay the contents of these new tabs can be displayed side by side with the graph The Protocol tab allows for monitoring assay progress while viewing the graph However to edit the protocol during the assay the Run screen must be used Tab Controls on Results Screen The Settings tab provides access to the rate calculation settings Typically a user might adjust these settings while o aa analyzing the data afte
123. ndicating the range E G 2 a oe oO s 40 60 TIME min 134 Deahorse Bioscience Threshold when graphing an analyte vs time Working with Multiple Data Sets The ability to view data from multiple data files in the same graph is a key new feature of the Data Viewer The Data Files tab as shown below shows the contents of the Data Viewer At minimum the original assay saved to this file will be listed To the right an Add button is available to browse for and load the contents of other data files The Delete button will remove data from the file The Clear List button will remove all added data The original data cannot be deleted or cleared Grid amp Graph Controls Data Files special Operations File Name MI FAD 07 13 07 16 11 49 FAD XF24 ASSAY DATA 1 xls Add Delete Clear List The Data Files tab To add data from another file Open your file 1 Click on the Add button Pe Menn 2 Browse for and select either an XLS or XFD data file Configure the browsing window to display only one file type by setting the Files of type option 3 Once a file has been k File name l Files of type ExcelFies e selected click Open Open dialog The list in the Data Files tab will update to indicate the contents from the selected file have been added 135 Aeahorse Bioscience Grid amp Graph Controls Data Files Special Operations File Name 07
124. nected paths of the empty bottle sensors to the vacuum intake port Install the vacuum filter blue on the vacuum intake see diagram to right Insert the large stopper Waste Hose Assembly into the large white waste bottle and place next to the PS Finally place the XF Plate Tray onto the PS as shown in the figure to the right Ensure that the rinse basin is toward the rear of the instrument and that the tray is square and level with the guidance tabs on the bottom aligned properly onto the tray track iil Tray Plate in PS 26 Aeahorse Bioscience Testing the Prep Station Connect the USB cable to the serial adaptor Connect the USB end of the assembly to the XF controller in the designated port see pages 20 21 and the serial end to the PS Place an XF24 cell plate into the plate holder Turn on the power of the instrument PS and the XF controller see page 28 Check that temperature displays are on and the bottle empty indicators are lit Note that they are only lit with empty bottles nearly empty bottles or when no boitles are installed Fill all bottles with water and check that the bottle empty indicators are now off To ensure that the hoses and valves are assembled correctly and connected to the proper bottles add a few drops EE of food coloring to each of the bottles use a different color for each bottle Launch the XF24 Software and log in see page 31 Click on the Plat
125. ned The graphs in the figure below show the results of pressing the f gt auto scale button before and after enabling this option To enable it 1 Click on the hy button to open the graph settings dialog 2 Enable the option to Match Y2 zero to Y1 This feature is under the Advanced Graph Options in the XF Reader Software 3 Click Close 4 Click on the gt A button 123 Deahorse Bioscience Lo Am re EVA Pa a Lia alha gt On the left Autoscaling with Match Y2 zero to Y1 disabled On the right Autoscaling with the option enabled Re sizing Graphing Elements The size of the font thickness of lines and size of data points can be adjusted individually To modify these settings 1 Click on the iw button to open the graph settings dialog and then on Advanced Options 2 For Font Size for Graph Line Width and Point Size choose either Small Medium or Large Displaying the Assay Date The date on which the assay was performed can be displayed above the graph To display it as shown in the figure to the right 1 Click on the iw button to open the graph settings dialog and then on Advanced Graph Options 2 Enable the option to Display Date in Title Graph with Assay Date displayed 124 D Jo gt pA lp gt f e gt eahorse Bioscience Displaying a Custom Graph Title A customized title can be added to the
126. normalized to cell count or some other factor then the AUC is calculated using the normalized data 115 A eahorse Bioscience ANOVA AUC Analysis Another AUC option is AUC ANOVA Similar to the AUC button the AUC ANOVA tool will EA Gia amp Graph Controls Data Files Special serations add a new worksheet to Group Sais gt AUC Anova Anyi Sa Harmatention ent the file The area under ses the curve is calculated for each group and then the groups ALE KEEN nata ewer ay Congas Lat bats Rat bata ate Wa Hate ats Tm ves Cate Eno Lag compared using ANOVA resulting in a table of P values Why ANOVA ANOVA provides a more rigorous statistical method for comparing three or more means The original AUC analysis tool uses multiple t tests to compare each pair of groups Using a single t test to compare two groups is valid But with more than two groups misleading P values can result when performing multiple t tests The more groups you compare the greater the chances of getting a significant P value by coincidence The new tool performs a one way ANOVA which compares the variation among the group means with the variation from the subjects within each group The null hypothesis is that all populations have identical means and a significant P value indicates that one or more of the population means differ from the rest To determine which groups are significantly different from the others the Tukey met
127. ns and features including customizing graph axes threshold options error bar format Baseline options Time Zero options and Background Correction Advanced Graph Options x xX TIME Miwa Vee i fo Font Size Line Width Point Size Maximum Vat 30 750 Small i Small f Small Mum Dinions fa fis 3 Medium Medium Medium eye o Large Large Large Threshold E ERK mo Graph Title Unt rm Moes l Display a in Title Show Ena Be YES lt fes ALEAR puto Scale Grid None Solid Ena Baw Format Staredaed Dansen Zero Marker Lines Rate Data Dirglay As MiddePowe Tine zo Pohi EOT W Draw Injection Markers Injection Markers Options C OterPort 5 Male2MnOsec Use Bar Graph when GROUP is on axis M Show graph legend when in well group mode to Seres Link Y1 of graph to daplsy va abtes I Ditplay Theeuhold on gach and on Do Rackgound Comecton themebeaks D Match V2 zmo to Y1 Basnine Subtiacton Methods Legend position Top Left Aseay NEURONAL BEP 090708 Pore t Poire 2 Port Point 4 Legend mode Colors foc Se el se OF Cancel x j es ae E Advanced Graph Options Graph Options Dialogue ee sub menu There is a sub menu that may be accessed via the Advanced graph options button that offers more options for customizing the graph including Font size Line width Point size graph title Grids custom Zero Time Point Injection marker options
128. nsion cable to one of the USB ports on the controller to allow for easy access to the USB port for data transfer to a USB memory device 3 Connect external network cables The controller may be networked via the Ethernet port on the controller 4 Connect the Mouse and Keyboard Open the mouse and keyboard set and insert the batteries properly Insert the wireless USB module into one of the USB ports on the controller Note the mouse and keyboard should function properly without inserting the accompanying CD into the controller Note that you may be required to install the Keyspan USB Serial Adapter software in order for the controller and PS to communicate with each other Please see below for detailed instructions on installation and set up of the Keyspan adapter Keyspan USB Serial Adapter Installation and Setup The Keyspan USB serial adapter is designed to connect the Plate Preparation station to the controller or separate computer It is a communications pass through that is required for the Plate Preparation station to be operated by a computer 20 at ty Se Deahorse Bioscience Installation Install the drivers for the Keyspan adapter BEFORE plugging the cord into the computer 1 2 3 4 Insert the CD with the Keyspan label that came with the instrument At the prompt follow the installation instructions If you do not have a CD with the drivers you can download the driver here http
129. nt scheme representing the spectrum of data values for each parameter Example Assay Results Non well group mode 56 A eahorse Bioscience Using the graphing buttons Me M D Graph Axis Drop Down Menus Use the drop down menus below the graph to specify which data is to be plotted on which axis OCR ECAR PPR OCR ECAR O2 pH or Note only the X axis has options for Time and Group When selected the group curve shows the difference in rates between the well groupings The x axis tick marks are labeled with each group name as specified in your assay configuration The groups can represent injected compounds treatment dosage cell type or any other factor 2 Pans the X axis to the right or left Expands or contracts the X axis of the graph Auto scale button fits data to the graph window Zoom Feature engages the zoom feature of the data viewer The Zoom Feature opens a window displaying the graph in a larger viewing format Note that Y axis now has panning expanding and contracting features similar to the X axis Also note the tab in the upper left of the window that allows the user to toggle between the larger graph view and a larger plate view All functions in the Zoom Window are the same as in the normal viewing mode see figures below 57 Aeahorse Bioscience Paama a 4 Zoom Mode Grid Zoom Mode Graph Graph Options Dialogue Button Opens a dialogue for multiple graphing optio
130. ntrol compounds The first injection is oligomycin Oligomycin is an inhibitor of ATP synthesis by blocking the proton channel of the Fo portion ATP synthase Complex V In mitochondrial research it is used to prevent state 3 phosphorylating respiration With cells it can be used to distinguish the percentage of O2 consumption devoted to ATP synthesis and the percentage of O2 consumption needed to overcome the natural proton leak across the inner mitochondrial membrane The second injection iS FCCP FCCP Carbonyl cyanide p trifluoromethoxyphenylhydrazone is an ionophore that is a mobile ion carrier It is an uncoupling agent because it disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase Complex V This collapse of the mitochondrial membrane potential leads to a rapid consumption of energy and oxygen without the generation of ATP In this case both OCR and ECAR will increase OCR due to uncoupling and ECAR as the cells attempt to maintain their energy balance by using glycolysis to generate ATP FCCP treatment also provides information as the magnitude of the spare respiratory capacity of the cells In the third injection rotenone is added to the cells Rotenone is a mitochondrial inhibitor that prevents the transfer of electrons from the Fe S center in Complex to ubiquinone Coenzyme Q This inhibition of Complex prevents the potential energy
131. ode is automatically engaged see diagram to right This window will be displayed with a 30 second count down If cancel is selected the auto maintenance is delayed by 1 hour lf start is selected or if the count down reaches 0 the software will switch to the maintenance tab and a rinse and soak command is started When it is completed the software will return to the screen before the warning The auto maintenance feature will engage every 8 hours and is recommended Note that it is OK to allow the PS tubing and dHO and Clean bottles to run dry there will be no damage to the PS just ensure that all lines are primed before performing a media change washing the manifold with dH2O D ERR 1 Flushes the tubos mih maii Pimi Agie 4 7 Pushes the jubes wih disivtecian ied Aae2S I foak iha tubes foes O ee i Phos tha tubers wah watir iia Raa Ss Boah iha tubas for 1 Ew PS Maintenance Rinse amp Soak 12 After the final XF experiment for the day is complete the tubing and cap that goes directly to the media bottle should be cleaned by removing it rinsing with dH20O clean disinfectant solution and QdH2O 13 Should the user wish to perform PS maintenance manually click on the maintenance tab in the PS screen and choose the appropriate option for your needs Rinse and Soak Overnight Loop or Long Shutdown 47 A eahorse Bioscience 10 Loading the XF Sensor Cartridge with Compounds The XF24 Sensor
132. omed view at right This is also Lecar evident if one uses the grouping feature mm FCCP 12 5K to display the data figure below Note meee that for both basal and FCCP stimulated rates the ECAR value does not increase significantly from 25K to 50K cells This indicates that the ECAR signal has reached a plateau somewhere between 25 K and 50 K cells well Thus using the 25K seeding number will allow one to observe both increases AND decreases in OCR and ECAR as this density is within the linear response range of the Zoomed view of basal ECAR vs Time cells used in this experiment ECAR mpH min 20 25 10 15 TIME min 012109 _C2C12 Blasts FCCP_Demo 012109 _C2C12Blasts_FCCP_Demo_1212009_ 1354 012109 _C2C12 Blasts_FCCP_Demo 012109_C2C12Blasts_FCCP_Demo_1212009_1354 Rate 3 Rate 4 OCR OCR 500 E OCR 450 wag ECAR 400 350 E 300 250 200 unu Hdw yywo3 OCR pMoles min uu Hdu ywo3 150 E 8 amp o oO oO FCCP 12 5K FCCP 25K FCCP 50K FCCP 12 5K ae FCCP GOK GROUP Basal OCR and ECAR Rate 3 FCCP stimulated OCR and ECAR Rate 4 Qualitatively one should expect similar responses as those shown in the graphs above lt is important to note that absolute values of OCR and ECAR may vary due to technical proficiency what type of and how many cells were seeded the degree of confluence at time of assay whether a consistent monolayer was obtained the exact pH of solutions and added
133. plate of C2C12 cells d Calibration buffer Seahorse Bioscience 2 Growth Medium a 500 mL DMEM Gibco 11965 092 b 10 FBS Hyclone SH90070 03 c 5 mL Penn Strep Gibco 15140 122 d 5 mL Sodium Pyruvate Sigma S8636 e 5 mL Glutamax Gibco 35050 061 3 Seeding Protocol a Cells are seeded in XF24 cell cultures plates with 25 000 cells well in 100 ul of growth medium and placed in 37 C incubator with 10 COs b Cells will adhere to the XF24 cell culture plate within 1 hour c After cells adhere add 150 ul of growth medium and return to 37 C incubator with 10 COs d Assay cells in XF24 24 hours after seeding 71 Deahorse Bioscience 4 Preparation of Assay Template Using the Assay Wizard Appendix l generate a template with the following group layout Column 1 3 BSA control Column 4 6 BSA PA Group 1 BSA Group 2 BSA PA Group 3 Temp Corr 5 Compound Preparation Prepare Palmitate BSA and BSA control according to the protocol in Appendix VI The palmitate preparation protocol yields a 1 mM stock which is used as a 5X stock solution 200 uM final 2 mL of a 5x stock is needed for the FAO assay described here 6 Cell Preparation and Media Change a Remove growth medium from each well leaving 50 ul of media b Wash cells with 1000 ul of pre warmed KHB running media remove 1000 ul as above and add 550 ul of KHB running media 600 ul final c Incubate in 37 C incubator without CO
134. pulling away from the PS e Remove the packing foam from underneath the manifold mount and remove the two thumb screws from the top of the manifold mount see right diagram below 22 Deahorse Bioscience e Unpack and install the manifold on the top of the manifold mount using the two thumb screws removed in the previous step The manifold is installed on the front underside of the mount with the metal nozzles facing down and the three plastic hose couplings facing towards the rear of the instrument Temp Display amp Empty Bottle Indicators O Thumb Screws l Spray Shield Please refer to list and pictures on pages 14 16 to assist with hose bottle and valve identification Use the diagrams below and pictures on the following pages as well as the diagrams on the PS itself for connecting the hoses valves and bottles Note that the hoses and connectors are coded with different color O rings to facilitate the connection process Often it is easier to install the hoses if the instrument is placed on the bench with the rear side facing out Thumb Screws 23 Seahorse Bioscience Bottle Hose Assembly Waste Hose Assembly Vacuum Filter Media hose Assembly Schematic Diagram of the PS Hose Connections rear view schematic Diagram of the Waste Hose Assembly and Media Hose Assembly to the manifold Top view Media Hose Assembly ito TOP nozzle of media pump Waste Hose _ Assemb
135. r Communications Settings Prep Station make sure that Comms is selected and that the light is GREEN lf none is displayed use the drop down to select Comm3 Click Connect After a moment the light should turn green 21 A eahorse Bioscience 5 Preparing and installing the Prep Station PS Unpacking the Prep Station PS with foam end PS in shipping carton PS accessory tray and boxes caps e Remove the accessory tray and accessory boxes from the shipping carton These contain the tubing valves bottles etc e Tip the shipping crate on its side and slide out the instrument and its foam end caps e Tip the instrument upright and remove the foam end caps e Place the instrument on the bench with the tie shield facing you see diagram to PS ready for packaging removal right Remove the tape holding the incubator door closed Open the incubator door and remove the packing foam Remove the plate shelf assembly and remove its packing sleeve Replace the plate shelf and close the incubator door Installing the PS Manifold and connecting the Hose Systems e Slide the incubator to the right of the instrument to clear the spray shield It will slide far enough so the left side of the incubator is in the center of the instrument see left diagram below e Loosen the two thumb screws on the left side of the instrument and remove the spray shield Lift the left side of the spray shield while
136. r capacity and volume Even when using the same cells these differences could theoretically produce different ECARs The relationship between PPR and ECAR is highly non linear and BC increases exponentially as pH decreases Buffer capacity can be defined in the running media definition of the assay configuration In addition a default value can be specified if running media is left undefined Two options are available for calculating PPR Variable and Fixed These options refer to how buffer capacity is treated in the calculation Variable vs Fixed The Variable technique assumes that buffer capacity will change appreciably during assay measurements An exponential function is used to calculate buffer capacity at 107 a ite eahorse Bioscience any measured pH level This function is derived from a table of pH values and their corresponding buffer capacity The pH values are in 0 1 pH increments starting at 7 4 The buffer capacity values in the table are initially calculated assuming zero buffer The actual buffer capacity at pH 7 4 is specified in the assay configuration as part of the running media definition With this value as a reference the ratio of the reference and zero buffer values at pH 7 4 is then used to scale the buffer capacity at the subsequent OH levels During an assay the exponential function derived from this table is used to calculate the buffer capacity at any measured pH level Knowing the well volume total nu
137. r the assay has completed see Appendix II Do background correction To make changes to a currently loaded Rates calculation settings data file the Settings tab in the Results ee J reece Pp TRF EGGE 7 Se004 Volume T screen must be used Changes made to l the Settings tab on the Run screen are Settings for ocr v not applied to the currently loaded data Those changes will only apply to the eai ake Cont avs v next assay During an assay the settings tab on the Run screen is not accessible Changes can only be made to the Settings tab on the Results screen Settings Tab on Results Screen Viewing results and basic graphing and analysis features Note that XF result files will be automatically saved as a MS Excel xls file that may be opened using MS Excel The following pages focus on the Excel presentation of the data Note there are 7 tabs along the bottom A brief description of each follows Data Viewer Displays the plate layout and graphic presentation of the XF results Features will be discussed in detail below 52 Deahorse Bioscience Assay Configuration exported from the XF Assay Wizard This tab contains all the information about experiment that was entered into the Assay Wizard Cell media compound info groups and labels protocol etc Level Data the raw data from the experiment in terms of pH and partial pressure Os This data cannot be changed manipulated or otherwise corrupted
138. ral Tab This is to input project info such as assay name project name etc Add desired information into the tab The Result File Name will be the save name for the file This name cannot contain any illegal characters If illegal characters are present the file will not be saved properly and will need to be manually saved after completion of the run Cells Tab This is to input or select info about cell type growth media plating info and cell layout 85 Aeahorse Bioscience Growth media To document Information about the growth media used It allows one to track lot numbers dates of preparation etc Note that several media compositions may be listed here Add or delete medias by using the add media or delete media buttons respectively The add media button will bring up a dialogue allowing the user to choose to create a new growth media or load from database Once the information of a growth media is added you may press the Save to Database button to log this information into the database Note this button will flash green to show that the information is added You may also add new growth medias directly into the data base by pressing new if the load from database button is selected j Berii Tut Ben Wasi Assay Wizard Cells Growth Media Screen Cells Info Similar to growth media tab above It allows one to track lot numbers dates of preparation etc Note that several cell types may be listed here
139. ral standard file and folder management features They all behave similarly to their standard Windows counterparts See Appendix V for more information about the menu bar and file management Bottom Row of Buttons right side Open Assay Wizard and Instrument Set up Open button prompts user to open a template or data file similar to Files button above Note templates and files may be opened with the open button or by double clicking on the template file itself Assay Wizard Used to generate new assay templates or modify existing templates for assay design A thorough discussion of the Assay Wizard is presented in Appendix I Instrument Interface for instrument set up preferences troubleshooting and Seahorse instrument and software maintenance Prep Station provides a series of screen for interface and operation of the Prep Station The Instrument Set up commands 35 eahorse Bioscience Lispa Optee O2 Levelt Display preferences defines ot EMV oom user preferences for units and graph options Units are relatively self explanatory note that some fields are fixed at this time od icy 1 od ba eee et be ap Met Te Display Preferences Screen The Graph Options screen fe is ipa siha OE s eerie eo Dane Mari maT Graph Options Main Screen The Graph Options Sub Menu This dialogue allows for user defined graphing preferences including preferred error display
140. re is only supported in the Data Viewer and is not available in the XFReader24 application Note that one may also explicitly define the X and Y axes with respect to minimal and maximal values number of divisions and sub divisions number of decimal places units and error bar options These may be found in the Graph options dialogue see figure to right s i Rate Data Display As Point to Point Rates v All Points Time Zero Point MiddlePoint First Recorda AA saat udi Graph Options Dialogue Point to Point Rates mode 130 at ty Se Deahorse Bioscience Adding Custom Text Labels to the Graph There is an additional new option on the right click popup menu for the graph object that allows for the addition of custom text labels to the graph s display To create or edit graph labels 1 Right click on the graph and select Edit Graph Labels from the popup menu The Labels dialog will appear as shown in the figure to the right Click on the Add Label button to add a new label In the Label Info dialog enter in the text for the label Also select a color and size for the text font Click OK when finished After all labels have been added click OK on the Labels dialog Add Label OF Cancel Labels dialog for the Graph Label Info Label This i a Graph of Font size C Small C Medium Large Cancel Label Info dialog
141. re they in an acceptable range lt 20 or are the errors larger Is there an individual well or wells that show deviation from the rest of the group e g obvious outliers that might be due to errors in seeding cells or changing media This may be facilitated by disengaging the average function and looking at the OCR and ECAR by group or by individual well or turning off the well group mode Note that this may be easier by deselecting the wells and or entire groups that you are not reviewing Intermediate Analysis Once you have reviewed the basic parameters described above proceed to an intermediate analysis if needed Baseline Function Recall that this baselines the data to a user defined point typically the measurement just before a compound injection and presents the data and graph in a format This is useful for knowing the fold enhancement or decrease in OCR and or ECAR PPR in response to an injected compound or chronic drug treatment For example the change in OCR may be viewed instead of the absolute rates This is useful when comparing data from multiple plates or experiments run at different times see below Absolute OCR Baselined OCR change 61 Aeahorse Bioscience Normalization Function To use the normalization tool some method to normalize cell count or cell activity must be used typically after the XF assay has been performed This could be an assay to ascertain cell number total
142. reen as described on page 2 and see diagram to right After Clean and dH2O lines tubes are primed click on the Media Change Tab The screen should look similar to the screen diagram on the right bottom Engage the Do Prime and Do Rinse buttons green set the desired final volume typically 500 1000 ul and be sure that ALL appears in the Selected Columns bar Place the cell plate into the PS and ensure proper positioning Press the green Start button The PS will prime the manifold with XF Assay Media then perform one aspiration and dispense cycle There will be a pause in the operation then the PS will perform a second aspiration and dispense cycle Do not remove the cell plate until all 2 cycles are complete z bas z are a EY A Aeahorse Bioscience 8 Remove the cell plate and place in the incubator until calibration of the sensor cartridge is complete 9 After the 2 wash dispense cycle is complete the PS will automatically enter into a self cleaning mode that consists of Clean disinfectant solution and dH20 again 10 If more XF assays are to be performed h simply repeat steps 4 7 above ensuring that adequate volumes of media dH20 and clean disinfectant solution are in the appropriate bottles Note that the PS will enter into auto maintenance mode 8 hours after the most recent use of the PS There will be a warning before the maintenance m
143. rt 3X J Mx 3 J Wat 2 J Measure 3 En 9 Expected results and discussion The purpose of this assay is to show the effects of cell seeding density on the basal and perturbed OCR and ECAR values As discussed above optimization of cell seeding number should be the first experiment performed with any cell line being used in the XF system One must be sure that the OCR and ECAR values are sufficiently above the background noise and within the linear response range of the cells Below are typical results obtained 012109_C2C12 Blasts_FCCP_Demo 012109_C2C12Blasts_FCCP_Demo_1212009_1354 012109_C2C12 Blasts_FCCP_Demo 012109_C2C12Blasts_FCCP_Demo_1212009_1354 Qocr _ Ecar 500 30 Gg C 12 5K Gg C 12 5K 450 JEE FCCP 12 5K EE FCCP 12 5K 70 EE C 25K EE C 25K 400 pete ES E FCCP 25K E FCCP 25K 350 JE c 50K 60 Jm c 50K EE FCCP 50K 50 E FCCP 50K 300 250 40 200 iS 150 i G 4 o 4 ECAR mpH min E 8 amp a oO 20 30 20 30 TIME min TIME min OCR vs Time ECAR vs Time Here we observe fairly linear increases with basal OCR values as cell density increases The ECAR data is less easily read but if we focus on the control groups during the first three measurements we see that ECAR increases from 12 5 K to 25 K 69 Deahorse Bioscience cells bu t begins to level O ff at 5 OK cells 012109_C2C12 Blasts_FCCP_Demo 012109_C2C12Blasts_FCCP_Demo_1212009_1354 Zo
144. s In the XF Reader software versions 1 3 and 1 4 the oxygen consumption rate OCR is calculated using the Fixed Delta technique for determining the slope The parameters Edge Offset and Delta define the boundaries of the data set used to calculate OCR Edge Offset defines the number of data points omitted from the beginning of the measurement period Delta set in mmHg defines the range of values to use from the starting point Seahorse Bioscience recommended that the Edge Offset equal be set to 1 and Delta to 50 mmHg in order to achieve the most accurate measurements These are the default settings in the software 98 Deahorse Bioscience Dual Grid Graph Protocol Do background correction 02 vs TIME Rates calculation settings 02 Buffer Capacity Volume 160 Ed Off i 1 e set Settings for v aaa RPE erT ite Cree Cee Cee re CeCe Cr ee Technique FixDeta Vv 5 140 130 120 Delta 50 O 440 42 0 42 5 43 0 43 5 44 0 44 5 45 0 TIME min Settings for Gain Fixed Method Improved Level Data Graph When looking at rate data and level data it is not clear where in the measurement the rate was calculated Depending on the rate calculation parameters some amount of level data Is typically omitted from the calculation 99 Aeahorse Bioscience The graphs of the level data will 02 vs TIME now consist of solid and outlined points The solid points will indicate those that are used to calculate th
145. s TIME Avg obtain information about the OCR basal respiration of the cells 1300 ee i in FCCP the percent of O2 consumption aoe jia iii devoted to ATP production as _ 1 an well as the amount devoted to 900 maintaining the proton gradient G 700 due to H leak Further we Respiratory may obtain the maximal 500 Capacity respiratory rate under p Basai Aana ri O Resp urnover conditions of uncoupled rr respiration sometimes 100 Leak referred to as spare respiratory Pr Y O capacity and finally we can 10 20 30 40 50 60 70 80 90 100 110 120 TIME min determine the amount of Os Respiration parameters consumption not due to mitochondrial processes 83 Aeahorse Bioscience As mentioned above this experiment can provide a framework for more complex experiments For example one may compare bioenergetic profiles from WT and KO cell lines with respect to the parameters outlined above Another example is an experiment in which cells are pre treated with a compound or drug of interest and asking how the basal and maximal respiration are affected A few suggestions before performing this experiment All compounds added Oligomycin FCCP and Rotenone or Antimycin A should be optimized for the concentration that provides the maximal effect That is one must perform separate titration experiments to ascertain these values This is especially important with FCCP as the titr
146. se Push button to the right and gently pull cover forward to remove 3 Calibration Measurement Position Used for SHB Utility plate during instrument calibration or cell plate during assay runs 4 Injection Manifold Uses compressed air to inject compounds loaded into Sensor Cartridge ports 5 Optic Fiber Bundles 6 Front and Back Solenoids Used for sensor Cartridge removal at the end of assay runs 7 Replaceable Plastic Tray Holds cell plate and utility plate with heat sink 8 Bar Code Reader Reads bar code on cell plate and back of Sensor Cartridge 9 Bar Code Illumination 10 Light Shield Blocks Opto Electric Compartment 11 Circulating Fan stabilizes temperature inside XF24 18 b Peahorse Bioscience Setup and Interconnects Cable Installation The XF24 Analyzer is operated from a touch screen computer mounted to a stand that fits up against the front of the instrument Two RS 232 cables handle the communication of commands and data between the instrument and the computer The controller may be connected to an external network A mouse and keyboard have been provided to allow the use of software other than the XF24 instrument control application Refer to the list on pages 13 16 to identify each cord and the diagrams on the right side of this page to identify the sockets z 2a a nuanco y b m 1 Connect power cords One power cord is used to on pee the instrumen
147. se for higher rates above 500 omoles min and changes as the gradient become stable Also increasing the delta or the drop across which we measure the slope provides more data points to calculate the rate Additional information on how to apply different OCR calculations to your existing data files can be found in the next section Instructions for Modifying Existing Data Files Modifying Existing Data Files The following procedure shows how to apply different OCR calculation parameters to your existing data files Note you will only see these OCR calculation options in the Settings tab if the data was captured using XF Reader software versions 1 3 or 1 4 If the data is captured using the 1 7 software version the Level Direct AKOS algorithm is the default setting will be applied to the data see next page for details 1 In the Files screen browse for and load an XLS or XFD ee eee data file display XFD files by changing the file display filter in the lower left corner of the Files screen After loading the file go to the Rates calculation settings Results screen to confirm the data was loaded Buffer Capacity eoa OE 2 In the Run screen select mee 6 foc D the Settings tab in the left ena as hand panel see Figure at wae FixDetta v right S O E 3 Display the settings for O2 If necessary click on the down arrow to switch from OH to O2 Click on the Edge Offset and Delta fields to modify their values
148. sure the pH of the running media and injected compounds are equal Again failure of pH measurements to return to the ambient pH of the media after each rate measurement would indicate a potential problem likely an inadequate amount of time for the sensors to re equilibrate during the mix and wait cycles Below are subtle and extreme examples left and right respectively showing the pH of the injected compound altering the pH of the media in the well Effects of Compounds on C2C12 Myoblasts PH vs TIME PH vs TIME 40 0 50 0 i 40 0 TIME min TIME min 65 2 at ty Se Deahorse Bioscience Sample Protocols for the XF24 analyzer This section contains the following experimental protocols that have been optimized by Seahorse Bioscience for data reproducibility and consistency Cell Titration and Uncoupling Experiment using C2C12 myoblast cells In this experiment 3 different densities of C2C12 cells are seeded FCCP is added to induce mitochondrial uncoupling which results in an increase in both OCR and ECAR Fatty Acid Oxidation FAO Experiment using C2C12 myoblast cells This experiment demonstrates addition of the fatty acid palmitate to increase the OCR of the cells Dose Response Experiment using HepG2 cells This experiment consists of injecting increasing concentrations of either DNP or FCCP and illustrates calculating an ECsp value for these compounds Bioenergetic Profile Experiment using C2C12 myoblast c
149. t at 4 C Preparation of 1x KHB assay medium Final composition of 1x KHB 111 mM On day of assay determine volume of medium needed from table below and prepare 1X KHB assay buffer as below Add indicated volumes of 5X KHB dH2O0 45 glucose solution and 50mM carnitine in a beaker containing a stir bar of 1x KHB dH2O 5X KHB a glucose 50mM carnitine Needed a mL mL E 2 o t 2 ft 156 at ty Se Deahorse Bioscience Alternatively add dry glucose and carnitine to the 1x KHB as follows g 100 mL 0 008 Warm to 37 C before adjusting pH Measure pH with pH meter and adjust pH to 7 4 For 100mL 1x KHB adjustment will typically require 10 15 uL 1N HCI Store at 37 C until ready to use Stock compound preparation for injections Be sure to use fresh molecular biology grade DMSO for preparation of compounds Ensure that after the compound of interest is diluted in XF Assay media that the pH of the solution is 7 4 10 mg ml Oligomycin Resuspend 5 mg Oligomycin Sigma 04876 5mg in 0 5 ml of fresh molecular biology grade DMSO Dilute in XF Assay media to achieve desired stock concentration for loading into injection ports Typical working final concentrations are 0 1 1 0 ug ml Aliquot and store at 20 C 10 mM FCCP Dissolve 10 mg FCCP Sigma C2920 10mg in 3 93 ml of fresh molecular biology grade DMSO Dilute in XF Assay media to achieve desired s
150. t to a ca grounded AC mains outlet A second power cord is used to Controller Ports underside connect the controller power supply module to the AC supply The power supply module is then connected to the socket at the bottom of the controller lay the controller on a flat surface to expose the connectors Repeat this procedure to connect the PS using a power cord and power supply module XF24 Rear Panel 6 2 Connect the data cables One RS 232 cable connects the controller serial port to the analyzer socket labeled COM 1 A second cable serial to USB no separate adaptor connects the analyzer socket labeled COM 2 to the USB port on the PS Rear Panel 19 Deahorse Bioscience controller directly adjacent to the network Ethernet port This port must be used for proper functioning of the instrument and barcode reader see diagram to right A third cable RS232 serial cable with a separate USB adaptor USB Port for USB Port for connects the PS serial port to the Com 3 eee USB port Com 3 which is Prep Station plese directly below the Com 2 USB port on the controller This port must be used for proper Controller USB Ports for Com 2 functioning of the instrument and and Com 3 connections Prep Station see diagram to right Please see page 36 below for further information on communication settings among the controller instrument and PS if applicable Connect the USB exte
151. tatistics Feature The Group Statistics button is located in the Special Operations Tab in the Excel Data Viewer Grd amp Graph Controls Data Files Special Operations ocr ecaR PPR OcRIECAR Grid amp Graph Controla gata Files Special Operol AUC Analyeme Groep Fisika eof Change OF Cale Mothed Hoa b HhData Viewer i Aray Configuration Level Data f Rate Data Plate View f Rate Data Tre Event f Caibraten finer log Le n View of the Special Operation Tab Options Clicking on the button generates another tab in the spreadsheet labeled User Data This sheet lists the statistics associated with each group for rate measurement Listed is the Group Name Mean Standard Error or Standard Deviation which ever is preferred by the user Number of Wells in the group Rate Group the wells in the group and the compound s added For example the statistics sheet for the Bioenergetic Profile using C2C12 myoblasts OCR data would look like this 111 eahorse Bioscience Group StatisticsOCR 090122 Bioenergetic_Profile C Documents and Settings grogers Desktop 090122 Bioenergetic_Profile MF PK_1222009 952 xls Project Name Assay Name User Manipulated Data Well Group Averages Experiment Date Baseline Mode Off Normalization Mode Off Group Name Mean Standard Error Num Wells Rate Time Group Wells CTL 106 6256023 2 622111125 7 1 1 09 46 AM 1 C2012 C2012 C2012 C2012 C2012 C2C12 C2C12
152. ted compound information for all wells note ports A D may be different Desc Provides user information and details for the project assay and plating Protocol Lists the user defined protocol for calibration mix wait measure and injection commands Info Bookkeeping info for the user detailing cells medium compounds and consumables Note the fields under the consumables tab for the plate and cartridge will be automatically populated when the assay is run Settings Instrument settings for temperature correction buffer capacity and rate calculation settings for OCR and ECAR see Appendix Il for more detailed information on buffer capacity and rate calculation settings 34 b Peahorse Bioscience 2 The Status button shows the status of the assay in real time including the current command current temperature time remaining for the current step and for the entire assay You can position the dialog by touching and dragging it by the top bar Touch the Status button again to hide the dialog 3 The Results button is used to review and analyze data after an experiment this will be discussed in greater detail in Section 12 4 The Files button see diagram below Used to navigate the file menu to open close save rename and delete files Use the left side of the screen to select and open folders use the right side of the screen to select and open individual files A menu bar across the top of the Files screen provides seve
153. the desired well to select it and then click on the Set normalization cell count button at the lower left This brings up a text entry box Enter the value and click OK Repeat this for all the wells and then close the dialog Then click on the button on the well grid to view the normalized data Set normalization cell count Normalization count entry dialog lf normalization values have not been entered and the Normalization button is selected a warning message appears providing an option to edit the values Selecting Yes brings up the same entry dialog The warning message is shown below 55 Aeahorse Bioscience Normalization Some wells hawe a normalization value of zero Do you want to edit the walues Warning if Normalization values have not been added Basic Graphing Features and Options Analyte Buttons and Rate control Use the appropriate button to choose which ocr ecaR PPR data is displayed in the plate layout OCR gt ECAR PPR OCR ECAR Os or pH Use the scroll bar to change which rate measurement lt 0 lt data appears in the wells and group boxes Rate 1 Rate 2 etc OCR ECAR Well Group Mode Click on the Well Group Mode Button to engage the Well Groups and color scheme defined during template set up in the Assay Wizard If this box is not highlighted the plate layout will shift to a histogram style of data presentation See diagram to right with a colored gradie
154. tock concentration for loading into injection ports Typical working final concentrations are 0 1 5 0 uM Note that response to FCCP will be dependent in Assay media composition pyruvate glucose presence of BSA etc Aliquot and store at 20 C 157 2 at ty A Deahorse Bioscience 100 mM 2 4 dinitrophenol DNP Prepare a 100 mM solution by combining 18 4 mg 2 4 DNP Sigma D198501 with 1 0 ml of fresh molecular biology grade DMSO Dilute in XF Assay media to achieve desired stock concentration for loading into injection ports Typical working final concentrations are 10 100 uM pH to 7 4 using 0 1 M NaOH Hint DNP will change the pH of the media significantly Use a pH meter or narrow band testing strip to check pH Comparing the color of the DNP solution to the same volume of unbuffered DMEM media is not accurate for pH as the DNP is a bright yellow solution that alters the color of the media even at pH 7 4 Aliquot and store at 20 C 1 0 mM Rotenone Prepare a 50 mM solution by combining 19 7 mg Rotenone Sigma R8875 with 1 0 ml of fresh molecular biology grade DMSO Perform a 50X dilution in DMSO to achieve a concentration of 1 0 mM rotenone e g 20 ul 50 mM Rotenone 980 ul DMSO Dilute the 1 0 mM solution in XF Assay media to achieve desired stock concentration for loading into injection ports Typical working final concentrations are 0 1 1 0 uM Aliquot and store at 20 C
155. ton after User Manager entering Instrument setup mode Accessing User Manager 148 Deahorse Bioscience Current User Info Login Seahorse User 1 Edit User Info The User Manager displays the current user Click on Edit User Info to edit the current user settings To edit other users or add new users click on Edit User List Edit User List Apply Settings End Instrument Setup Mode User Manager When accessing the Edit User List function for the first time the Admin gt user password is required The Admin password initially defaults to ADMIN Guest This password as well as any user Seahorse User 1 password can be edited by selecting a user login and clicking on the Modify button When modifying any user can be elevated from a Normal User to an Administrator Only Administrator level logins can access this section To remove unneeded users select a user login and click on the Delete button New user New users can also be created here Cancel instead of at the main login screen User Manager 149 Aeahorse Bioscience Refresh File Menu and File Filter A Refresh bution is located on the Files screen to update the list of available drives In prior releases the drive letter list was populated when starting the software and could only be updated by exiting and restarting After plugging in a USB flash drive to the instrument controller or any other computer runnin
156. ton in the right panel 3 Inthe left panel select Database 4 For each database file select the desired file location When finished click on Apply Settings and End Instrument Setup Mode Suggested Workflow Simplify the creation of assay configurations by using the database feature to store definitions or partial definitions of frequently used assay materials For example 1 Load a partially completed assay configuration template into the Assay Wizard using the Load from Template bution in the General tab 2 Navigate to uncompleted sections of the assay configuration load definitions and perform layout assignments At the end of the wizard use the Save Template button to save the completed assay configuration to an excel file that can be copied to the touch screen controller If making these modifications on the touch screen controller end the wizard and proceed directly to starting the assay User Login and Profile Preferences Overview User logins and passwords can now be used to access the XF software The new Login screen shown here in the figure below is displayed when first starting the software Various software settings will be associated with each login including default assay configurations and folder locations Each user login can also be configured with a default starting screen such as the Results screen or Run screen A Guest login requiring no password is provided Editing or removing user logins can b
157. undaries at various rates that are determined by the Chamber Oo magnitude of the gradient and the physical properties of the boundary O2 w walls In addition these boundaries act as storage compartments for oxygen and therefore the functional volume of the trapped measurement chamber is larger than the actual physical volume of 7 ul for the XF24 V7 cell culture plates Ambient The new algorithm accounts for the flow of oxygen across these boundaries by using Oxygen Sources a set of defined rate constants To correct for the difference between the functional and actual volume of the well chamber a volume of 22 7 ul is used for the OCR calculation Researchers at the Buck Institute for Age Research were integral in the development of this new algorithm and it was validated by comparison to classical Clarke electrode oxygraph measurements For more details you can contact Seahorse Bioscience and request a copy of the journal article that details this research Akos Gerencser et a Analytical Chemistry 2009 Applying the New Algorithm to existing Data Files Although you can apply the new algorithm to your existing data files OCR data in the recommended range a total drop in partial pressure of O2 lt 50 mmHg will be relatively unchanged OCR data outside the range can be rescued It is important to note that OCR data gathered with prior versions of the XF software was collected accurately The method
158. use included may also be used The controller communicates with the analyzer using a single digital communication RS232 cable and a single Serial gt USB cable and communicates with the PS via a single Serial gt USB cable Plate Preparation Station PS optional The PS is optional but recommended for exchanging growth media for assay media in the tissue culture plate wells It also contains a temperature controlled oven for incubating the cell culture plates in a 37 C non COs environment after the media exchange and a temperature controlled dry bath for warming and incubating the assay media Prep Station PS and XF24 Flux Analyzer The XF24 and Prep Station require two people to lift and handle safely Each person should firmly grasp the base of the unit at opposite ends to one another Use OSHA standards for lifting techniques 1 e eahorse Bioscience The following items are included in the XF24 instrument and controller boxes XF24 Instrument XF Controller XF Controller Stand Power Cord instrument Power Cord controller Power Supply controller RS232 Cable 13 A eahorse Bioscience RS232 gt USB Cable Wireless Mouse Keyboard USB Extension Cable Extra Fan Filters Excel 2007 CD USB drive Prep Station Prep Station 4 channel Manifold 14 Deahorse Bioscience XF Plate Tray Power Cord RS232 Cable RS232 gt USB adaptor and USB cable Reagent
159. ve portion of the graph Oxygen Level Data Calculated with New Algorithm 103 lt eahorse Bioscience Here is the OCR data using the original algorithm n 2 i amp a Oo re Here is the OCR data with the new algorithm Note that the OCR values have not appreciably changed OCR pMoles min Additional Benefits of the AKOS algorithm Point to Point rate display mode Another feature of the AKOS algorithm is the ability to view the rate measurements in a point to point mode since the algorithm is able deconvolute the instantaneous OCR during the measurement the user has the option of displaying the data as the middle point or as point to point rates throughout the measurement period The middle and point to point modes can be accessed via the graph options dialogue box see diagram to right Only the xls data files support the point to point mode Num Decimals fo 0 Threshold 250 Range 10 Unit min pMoles min Show Error Bar YES X NO Auto Scale x Auto Scale Y1 Bar Format STS EAO C Other Point 4 1 Mix for 0 Min 25 sec Link Y1 of graph to display variables Display Threshold on graph and grid V Do Background Correction Apply threshold to Series Match Y2 zero to Y1 Baseline Subtraction Methods Assay ISO MITO Point 1 Point 2 Point 3 Point 4 C User Defined Point From Injection Point V A B E
160. ze yourself with these cell culture techniques XF Assay Day 1 6 Seeding Cells in XF24 Cell Culture Plates Assays are performed in a Seahorse ls oe S a 24 well cell culture plate in conjunction le S a with an XF24 sensor cartridge Both d RAE s are formatted in a typical 24 well format with rows designated A D and columns 1 6 The seeding surface of each well is equal to that of a typical 96 well plate XF24Cell Culture Plate 42 at ty Se Deahorse Bioscience A two step seeding process is strongly recommended when seeding XF24 cell culture plates This procedure produces consistent and even monolayers of cells 100 ul cells 150 ul medium 1 5 hours 37 C CO incubator A Resuspend cells to obtain desired final concentration to seed in 100 uL of growth media Typical seeding densities vary from 20 000 to 60 000 cells per well B Seed 100 uL of cell suspension per well leaving the appropriate background correction wells unseeded A1 B4 C3 D6 Be sure to put media only no cells in the background correction wells Hint Hold the pipette tip at an angle and rest the tip just below the circular rim at the top of the well for best technique and homogenous cell layer C Place plates in incubator and allow cells to adhere This generally takes approximately 1 hour for strongly adherent cells but may take up to 5 6 hours for less adherent cell types D After cells have adhered add

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