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Wave User's Guide

1. 53 Access to Predefined Conditions 1Leeeee eee ee eese eee eee eene eene enne enne tnnt nnt not no neun 53 Customizable Default Settings usi err ire ea erre uerk Ese aus or xo eR a pes Cv Es ES a E RE CKeS cs eae n Des VuUPEEeUN EE Ue Ee Es 54 e cWr om Y 54 BUNE DAC EY EROR 55 Atmospheric E51 EET NS 59 Recent LIS cc r 55 DTIC NSS RR E 56 FA ONG i um zie 56 Wave User s Guide VS CRU IO ys vie vag esas acter M M 56 Protocol Defaults cT 57 PQ 58 PON INC SN isis etc NA A A A weer ec creas 58 Chapter 4 Analyzing Your ASSAY E K ESENE E Sa 60 Arrows for Expanding a Graph or Chart 60 Zoom Pan and Restore Buttons 1 ee ee en ea eo se ae aepo ne tesa et ee nai reae oae eoa soon oaa a a aaa ae Eo seek aeos naue 61 Analysis OVEIWIOW S cicsicv lada RES IO RENE ER RISE ES E UFUR NIE EE UNE REVO 61 Minime Cie 2 NENNEN TEE omn 62 OCR ECAR OF PPR de E M M 63 Raw Level Daila M PE 63 OCR ECAR PPR 03 OF DH OVENAY 66eeeeeer 63 Standard Deviation and Standard Error of the Mean sssssssssssereerererersssssssssssssssrsessesees 64 Kinetic Graph Customi zation es rie nito pet rhe eb ea
2. 80 Append information from your assay sssssssseeeeeeeeennenennnnnnnn nennen nennen nnn enne sina annee nnns 81 la T He 84 dssdo 85 Analysis TOOL BUTTONS ER 86 Groups and Conditions 86 To edit groups and conditions 86 ASSaV 6 8 87 To modify the assay properties scicccccesssdsassiavedsnedaessacscnncdoceasessanseesensoosasovestwosaasdeavavacsaseens 87 ONIN eZ AE Ca E E 89 To copy and paste from a secondary SOULCE sssesssseecccccecessesssececeecesaueaeseceeeesesauaagsseeees 89 To enter normalization values manually 91 Analysis witli EXCela rec 92 To transfer the entire results file asyr to Excel for further analysis 92 To transfer data from a graph or chart to Excel for further analysis 93 Wave User s Guide Chapter 1 Designing Your Assay After a brief overview section on the entire process of designing running and analyzing assays this chapter explains all of the steps to designing an assay Introducing the XF Home page Starting a new assay in Start a new assay design Defining conditions starting with Assay Conditions Injections Creating groups in Automatically Generate Groups Mapping groups to the plate map in Map Gro
3. sees nennen nnne 32 Chapter 2 Reviewing and Running Your AsSay csccscsccscssccccecsscecceccsceccnccsceccsccececcscecesens 33 Log onto the Wave SOTEWaL Gone oed a VR aEESC RE THES VE TRESR CUR EEPETXAR YER MM a VITATE S9G Ce bRO FER KS Yu Ewa MAY VE QE YA EOd 33 Open d Desien File c os AA nA rU DC EISE DUE 33 Opena Template File sias ovs RASo dU TAPA C rA IINE N SAFE e PIRA dS 34 Review the Assay Design iiis sra kunt aine ua a PYFRNTEERESNRRRYWARVARERENAxA nV Ee kNauERA KY aOV ae kada ER EXE Saa Vae uu ae VERE S 35 2 Wave User s Guide Review the Design of Your ASSAaY ccscsscsccscssceccsccnceccncccceccsccececcsccccecssseccssesceccnsscescsseeceees 36 General INFORM ALON RENTE TEL mm 37 Email Recipient B RERO RTT 37 Advanced Opon cR TET 37 d EE REEE 38 OUI senses A A E E 39 mmm 40 E A E EEEE E N A E E EE E 40 Run YOUN ASSAY oia E 43 Temperature Control 44 E T AAA E A 46 Prone CONTO PRONUM I 47 PI oE EO e 47 Cartridge barcode read failure 48 EAE 50 1 51 Chapter 3 Managing Your ASSayS iisccccsicecscissccconssiceccnnseceecsvaceecsanscentivedeccusppadeiceicexeiwseeeetivadees 52 Access Directories Analyses and DeSIgins ccscsscsccscssceccsccnceccscccceccscccecssceccecesceccnsenceccnss 52 My Places and My ASSayS ccccsccssssssssescecssccssssesesecccssccssessssecessucnsusesesssceseneueusssessscecsseseussesssss 52 ls um
4. 55 Wave User s Guide After specifying the maximum number under each setting click the Save button at the bottom of the screen Review Access to your directories analyses and designs to see how these default settings can affect your access to designs and analyses Directories The Directories column shows the default location of your Catalog directory and your Template Directory Directori Catalog Directory CAProgramDataXSeahorse Bioscience Template Directory C ProgramData Seahorse Bioscience If you would like to change the location of either of these directories click on the button to the right of the directory field to browse to the preferred directory or type the path name of the directory in the directory field After specifying a new directory click the Save button at the bottom of the screen Favorite Places The Favorite Places section near the bottom of the page is for adding or removing directories form the My Places screen nhorvath Documents My Assays C Wsers mbhorvath Documents Help To add a folder to the list of folders that Wave displays under My Places click the Add button to browse to the location of the folder on the Select Folder dialog box and then click the Select Folder button To remove a folder from the list of folders under My Places select the folder and press the Remove button After adding or removing a directory click the Save button at the bottom of the screen Inst
5. Comment MH1 Please give me a better definition In some cases it is useful to normalize your rate data using results from another assay or laboratory technique in order to report a response in relation to another measurement such as per cell or per quantity of protein You can do this by selecting Normalization in the tools button and adding or pasting in outside normalization data There are two ways to add normalization data to the Normalization window e Copying and pasting from a secondary source e Enter the data manually To copy and paste from a secondary source 1 Copythe secondary source data from a spreadsheet or similar grid format to the Clipboard by using the key sequence Ctrl C 2 Click the Modify button and select Normalization from the dropdown menu 89 90 Wave User s Guide HHH iti Hii Groups Conditions T THE Assay Properties ul Inie Normalization UU0 7 0 57 7 0D D05 25 2 amp k e Click the Select All This will select all of the wells in the grid E Paste Click the Paste Jbutton This will paste the data from the clipboard into the normalization grid Check the numbers in the grid to be sure they are correct You can also enter the data manually by selecting a well in the grid and typing in the value Type in a Normalization Unit in the Normalization field at the top of the screen This unit will then b
6. Append To Sum num To see what you have appended in your summary document 1 Click the Summary tab on the top right side of the screen For more information on editing your summary document see the Summary Tab section 66 Wave User s Guide Rate Details and Bar Graph The following sections explain how to understand and manipulate data in the following parts of the screen e Rate details e Bar graph e Group details Rate Details In the upper right hand corner of the Overview tab the Rate Details grid is displayed OCR EE 11 c E E E E E fo fm bo alee eT fede oo fom fom om fom lee Yom or fom low fom ow oom oe CN Here individual rate data points are displayed for each well at a specific measurement point To change the measurement point displayed in the Rate Details grid choose from one of the points in the Measurement dropdown menu which is on the top left hand side of the screen as shown in the following illustration on the next page 67 Wave User s Guide lg Fe 22 0 40 587 10 36 d UC XFe Cell Mito Stress EJ hd EA A Baseline The measurement point that you select will be highlighted in a blue column on the kinetic graph as a reminder of which rate data bar graph and group details are being displayed in the Rate Details grid If OCR is selected in the Rate dropdown menu your Wave software will disp
7. e Extracellular acidification rate ECAR e Proton production rate PPR The default view displays time on the X axis in minutes and rate on the Y axis You can change the displayed rate OCR ECAR or PPR by selecting it from the Rate dropdown menu above the graph If you select OCR the O consumption rate data pmol minute will be displayed If you select ECAR the extracellular acidification rate mpH minute will be displayed If you select PPR the proton production rate pmol minute is displayed Raw Level Data You can display the O or pH raw level data used to calculate the rates To view the raw level data select Level from the Y1 dropdown menu OCR ECAR PPR O or pH Overlay In some cases it is desirable to overlay two rates on the same graph You can overlay two rates on the same graph by selecting a different rate or measurement from the Y2 dropdown menu If you select a rate Wave will display the rate for the Y2 axis and the rate for the Y1 axis on the same kinetic graph so that you can compare them 63 Wave User s Guide If you select O Wave displays the O mmHg measurement level for each group The software will show the exact numeric measurement if you hover over the group line on the kinetic graph Similarly if you select pH Wave displays the pH measurement for each group Similarly the software will show the exact pH if you hover over the group line on the graph Y
8. 5 48 Minimum Sum 280 05 Sum 205 v 3 54284 11 81362 Group the Data by Field To group the data by field 1 Click on a column heading and drag it up to the gray bar In the following example the GroupName heading will be dragged to the gray bar GreupData Rate levelData Raw Calibration Calibration View Event Log Background Creme ces ey group by Areg Drag a feld here te gren by that held Mesturemenn ty Tene 13033 2a 1 LEJU QUEE PL 25K Cutie a 1 Pat SK DUDOOOO 1 Ad IUE QUOD i PC 3 20K 10177912 1 54548 DDow i PC ga ian 1 Ad Be aCi ia n El Barkgnumd IBATI Leos QUOD 2 POCO D 2 BONA 12155 a00000 ALTI 48350 00000 Cubo TORI The groupings will appear as shown in the following example 77 Wave User s Guide ES Cell density optimiz E Be s e ase view Summary Plate Ed Data View Ed To expand a group 1 Click on the plus sign to the left of a group to expand it After expanding a group you can select another column heading to define the groupings further In the following example the heading OCR is dragged to the grey bar after GroupName de Fe 200 BE x cet density optimi E EY sme aP adc view Summary Plate EJ Data view Ed Raw Calibration Calibration View Event Log I Greuphlame gt 1 rjncgm mq OOR Erra 1 ECAR LI ECAR Enter T PPR 17 Peg LOK TiLE y08 1577154 627735 651935 azo
9. Wave User s Guide The Open command provides a screen where you can view all your assay related files templates design files and analysis files Ba Templates A template is the framework for an experimental design file that has condition fields pre filled with definitions provided in a previous assay design The advantage of using templates is that you can start an XF assay quickly or maintain consistent record keeping information by modifying a previous assay design You can save any design file as a template Templates have the file extension asyt When you first open Wave you will see only one Blank template All the templates you create will appear in the Templates pane Each template that you define has an icone You can manage your templates by using the Import Export and Remove buttons below the Templates pane A 5 New Design EJ XF Home NEW ASSAY Instruments ln XFe24 XFe24 Hypoxia Mode dm xre26 XFe24 Analy XFe96 Ext XFe24 Extracellular Flux Analyzer yzer installed in a Hypoxia Chamber 296 Extracellular Flux Analyzer Open t Catalog Templates The person indicates that this template is user defined Options Glucose Test Removes templates you no longer need Moves a copy of a template to a different directory or a storage device Imports additional templates Eq Import Import Export and Remove Buttons e The Import button allows you to import a template fi
10. however if you intend to use two injections you will need 9 measurement cycles starting with the Basal Measurement Cycle The following table shows you the number of measurement cycles for 1 to 4 injection cycles Measurement Measurement Cycles required per Cycles per assay Injection Basal 3 3 Injection 1 3 6 Injection 2 3 9 Injection 3 3 12 Injection 4 3 15 Basal Measurement Cycle The Basal Measurement Cycle consists of the first three measurements that occur before the first injection This command is the default starting point for all XF assays Touch or click on the dropdown arrow on the Edit Measurement Details bar to change the default Mix Wait and Measure times The recommended Mix Wait and Measure times are instrument dependent you can set defaults in the Instrument Options tab see XF Home for more information 3 Measurement Cycles Mix Wait Meas 03 00 02 00 03 00 Edit Measurement Details Cycles Mix Wat Measure h A A te 03 00 02 00 03 00 27 Wave User s Guide If necessary use the up arrow a or the down arrow we to increase or decrease the number of Cycles for each measurement Seahorse Bioscience recommends 3 cycles per measurement for optimal results Change the name of the Basal Measurement Cycle by selecting the text field at the top of the column and typing in the desired information Injections Each time you select the Injection button a new Injection command will be
11. 3 0 81746 1 02424 l 0 00000 Group 4 0 81746 1 15717 0 06798 Group 4 0 81746 0 80503 24417 2 21258 Each data point is identified with a well column and a group column so that the data can be displayed in a number of different formats Sort by Column To sort the data by a specific column 1 Click on the column header Measurement Well Group Time OCR ECAR or PPR Wave will sort the data in ascending or descending order in the chosen column Display in Ascending or Descending Order To change from ascending to descending order click the arrow to the right of the column name In the following illustration Wave lists OCR data in ascending order You would click the up arrow to the right of the cursor to change to descending order Group Data Rate Level Data Raw Calibration Calibration View Event Log Background Correction Measurement Well 2 A09 T0564 1 38036 2 A05 4 0 00000 Display the Average Count Maximum Minimum or Sum To display the Average Count Maximum Minimum or Sum or any combination of these values click on the Sigma sign to the right of the arrow and check the applicable check boxes In the following example all of these values are checked for OCR data therefore Wave shows the average count maximum value minimum value and sum for the OCR data 76 Wave User s Guide Measurement Well Average 0 97 Count 288 Maximum 10 97 Maximum Minimum
12. 78 Wave User s Guide Export to Excel from any View You can also export the data to Microsoft Excel from this view To export data from here or from any analysis view 1 Right click anywhere on the graph for touch screen hold your finger on any part of the grid for three seconds Your Wave software displays the following menu Copy Export Print 2 Choose Export The Export to Excel dialog box is displayed ig Save Assay CO di My Documents XFe Assays hd t Organize New folder Yr Mame Date modified BE Desktop AFe Cell Mito Stress Test Data 8 26 201312 12 PM Microsoft d Downloads l Recent Places EE Desktop Libraries ES Documents al Music i My Assays Pictures BE Videos File name 2E BTE EN EET Save as type Excel 2007 2010 xlsx Hide Folders Browse to a directory where you would like to save the Excel file Type a name for your file in the File name field Select Excel as the type in the Save as type field Click the Save button E es Wave will export the data grid exactly as it appears in the data window so that you can perform further analysis in Excel 79 Wave User s Guide Summary Tab Edit In the Summary tab you can customize your summary and add graphs and charts to it Specifically you can do the following e Document a summary of the run complete with kinetic graphs bar charts and group and protocol definitions e Pri
13. NO Ovpicate h Gereme Groups are defined A o Injections nm Injection Condition i UC a a no pyruvate low fecp 7 Background pyruvate Pigh fer z A B C aD P no pyruvate low fccp Ns Pretresteents tro pyruvate iow fecp o No Meda 4 Compound x Pretreatments no pyruvate medium cep No Pretreat ets 3E XF Assay Medium no pyruvate emdim io Meda DO Cell Type pyruvate high fcp yruvate high fecp No Pr to Media pyruvate low tocp ate low fece to Medu pyruvate medium focp yoi wol Ko wo 4 Define injections for each port that you intend to use during the assay You can switch between ports A B C and D by clicking or touching on the tabs in the Injection Condition window If the experiment uses the same injection strategy for all wells and all 4 ports specify one injection strategy Assay Conditions Pretreatments Pretreatments describe any treatment the cells have received prior to the assay such as a genetic manipulation or a drug treatment To define pretreatments 1 Touch or click the Pretreatments bar s Add Pretreatment Condition 2 Click the Add Pretreatment button 20 Wave User s Guide 3 Click on Pretreatment 1 to display the Edit Pretreatment dialog box In the following example Pretreatment 1 has been renamed Growth Medium UY save Group Definitions Instrument Protocol Review and Run nditions Add Saxe Remove Duplicate E
14. PDF HP Color Dell Laser Printer 1710n on thmsrdcO12366 i HP Laser Printer status Ready Documents 0 Location At David Min Office Number of copies 1 Curent Page Collate 1 Select the printer that you would like to use 2 Select the pages that you would like to print 3 Select the number of copies that you would like to print 4 Press the Print button to print out the summary The Summary tab is always accessible when an analysis file is open This information is used to record how the assay was run mix wait and measure times along with injection sequences are shown as well as information about where the groups are located within the plate 84 Wave User s Guide Export The Export buttons allow you to export your summary document in 4 different formats e Word e PDF e HIML e Rich Text To export your summary document 1 Click the appropriate icon from the Export section of the tool bar ps DD Word PDF HTML Rich Text Organize New folder Ea Date modified X Favorites NE Desktop m Downloads Recent Places Mo items match your search y EE Desktop Libraries Eg Documents a Music tall My Assays l Pictures BE Videos File name Save as type Word Documents docx Hide Folders Browse to the location where you would like to save your summary document Type the name of your summary file in the File name field Select the type of file from the
15. Save as type dropdown list prox M9 Je Press the Save button to save your file 85 Wave User s Guide Analysis Tool Buttons In any analysis view you can make changes or additions to the data views to assist with more advanced types of analysis You can take the following actions e Edit groups and conditions after a run e Make changes to the assay properties recorded in the Review and Run section of the design file e Change the name of the injections e Apply normalization factors to rate data hye Modify The Modify button in the upper right hand corner of the Summary screen provides a menu of actions to take as shown in the following illustration TH Groups Conditions ISE Assay Properties OQ Injections Normalization Groups and Conditions To edit groups and conditions 1 Click the Modify button and select Groups Conditions from the dropdown list Wave displays a reproduction of the Conditions and Plate map windows found in the Assay Design section of the software m he 204050 B7 10 16 d iii xFe Cell Mito Stress EJ Ee XFe Cell Mito Stress EJ Groups Conditions Add E fiackgraurd B llackgreund Conditions Desenbe He No Media gt 8 ii gnyrorvade how boop o ew pyruvate Kev legge T o Ne Fretrean Do Ko Cal Typer H no pyruvair medium boop o Pus privat maior ow aem Plate Map No Media Ha Gen Types bees Rl che Plans pyruvate high feep 6 pyrev
16. Summary JT Mix 00 03 00 Wait 00 00 00 1 Measure 00 03 00 1f Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 Injection 4 Inject Port D YW Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 f Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 Injection 1 Inject Port A JT Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 Wave User s Guide Print Summary TOTAL TIME 01 06 00 Injection 2 Inject Port B JT Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 E Measure 00 03 00 The Group link allows you to review the groups that you have defined for your assay To change these group definitions 1 Click on the Group Definitions tab 2 Make any necessary changes to your conditions or groups prior to running your design Refer to Define Groups and Conditions for more information The following illustration shows an example of a group summary Groups Group n Background no pyruvate low fccp no pyruvate medium fccp no pyruvate medium fccp No Pretreatments Bl pyruvate high fccp n pyruvate low fccp pyruvate medium fccp a no pyruvate high fccp2 n Background BBBS SEBBRAOB DB EU BEBBEBEEB000U08 39 L7 Print Summary Q Injection Pretreatment 2 Media N A N A N A No Pretreatments No Media No Media no pyruv
17. also monitor your run and save your design file 35 Wave User s Guide The following illustration shows the purpose of each of the buttons and icons on the Review and Run window he Review and Run tab brings you to this window Prints your entire summary report including protocol and group summaries Saves your assay design eee XFe Cell Mito Stre EJ Group Definitions Instrument Protocol Review and Run GENERAL Information gh Prix Summary Project Information Plate Information Advanced Settings Project Name Well Volume ul Emal Notification Advanced Pyruvate metabolism 200 Principal Investigator Plated By Irons L Janes email address Add Project Number Plated On 01 8 26 2013 m orts A B C and D display a summary of njections to each of the ports Notes START RUN A Note The 7 e _ button is to the right of this window on the touch screen This chapter is divided into two parts that explain how to 1 Review the Design of Your Assay 2 Run Your Assay For more information on saving design and template files see Save and Save as Template Review the Design of Your Assay Prior to running your design you can check many aspects of the design e Review the General information that you see when you first open this window or click the Summary link on the left hand side of the screen e Click on the Protocol link to see Protocol information e Click on the
18. d basim ie Dasigns GlycalysisAngdstress Tests ny Pusgust 27 2013 4rd PM Cell denzuty optimization with ze mita stress Test Mew Keda Assay 33 Wave User s Guide 3 Click the Open Button and select the Open option from the dropdown menu to Open your design file O Jb Libraries Documents gt XFe Assays v Organize v New folder oe e Ft Favorites Documents library BE Desktop XFe Assays m Downloads E Recent Places Arrange by Folder v Name Date modified _ GlycolysisAndStressTests asyd 8 27 2013 4 00 PM BE Desktop 2 Cell density optimization with cell mito stress test 8 26 2013 1 20 PM A Libraries _ XFe Cell Mito Stress Test asyd 8 26 2013 1 19 PM E t _ Cell density optimization with cell mito stress test asyd 8 23 2013 3 18 PM a Music 2 GlycolysisAndStressTests 8 23 2013 310 PM il My Assays 2 mito glyco 8 23 2013 3 08 PM E Pictures _ New Xfe24 Assay asyd 8 20 2013 4 24 PM B Videos B Mary Horvath B AppData H Contacts k Desktop Filename Cell density optimization with cell mito stress test 4 Click on the Review and Run tab Open a Template File To open a template file asyt and prepare to review it 1 Click on the New command under XF Home 2 Ifthe template is not listed in the New Assay window click the Import Ibutton at the bottom of the screen to import your file 34 Wave User s Guide 3 Locate you
19. included in the assay protocol aJ Injection 1 EH see y EE oh y Measure after Injection 3 Measurement Cycles Mix Wait Meas 03 00 02 00 03 00 v Edit Measurement Details Group Summary For each new injection the software will automatically select the next available port You can change this default setting by selecting another port A B C or D Only available ports are selectable if a port is already assigned it will be greyed out Edit Measurement Details When you select an injection the software will automatically add a set of measurement cycles after the injection Default mix wait and measure times are provided however you can customize these times as well as the number of cycles If a measurement is not required following an injection you can uncheck the Measure after Injection box Group Summary Select the Group Summary bar to view the compounds assigned to these ports 28 v Edit Measurement Details Group Summary Port A Inj Strategy 1 No Pretreatments l insulin H20 Custom Sequence Wave User s Guide Most XF assays require only measurement and injection cycles but in some user defined assays a custom sequence of commands is required In these cases select the Custom button to add acustom sequence cycle to the assay Here you can add as many mix and wait cycles as your protocol requires Save and Save as Template After your assay design is complete y
20. the tool bar The following illustration shows an example of the General Information section that Wave pastes into the summary for you GENERAL Information Project Information Plate Information Advanced Settings Project Name Well Volume ul Email Notification Advanced Pyruvate metabolism 200 Principal Investigator Plated By J Irons L Janes Project Number 01 Plated On 0 6123 O o ZU 12 You can also add the Protocol summary To add the Protocol summary Eu Protocol 1 Click the Protocol button The following illustration shows an example of a protocol summary 81 PROTOCOL SUMMARY E Calibrate tet Equilibrate 00 00 00 JT Mix 00 03 00 J Wait 00 00 00 Measure 00 03 00 JT Mix 00 03 00 7 Wait 00 00 00 P Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 P Measure 00 03 00 Injection 3 Injection 4 Inject Port C Jf Mix 00 03 00 Wait 00 00 00 P Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 Measure 00 03 00 Inject Port D JT Mix 00 03 00 Wait 00 00 00 P Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 P Measure 00 03 00 You can also add the Group summary To add the Group summary 1 Click the Group button Injection 1 qm Inject Port A JT Mix 00 03 00 Wait 00 00 00 P Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 Measure 00 03 00 TOTAL TIME 01 06 00 Injection 2 Inject Port B Jf Mix 00 03 00 Wait 0
21. to a run after a run is complete or after a run has terminated due to an unexpected error it is sometimes necessary to raise or lower the probes or load or unload a cartridge If you need to raise or lower the probes or load or unload a cartridge U 1 Click on the Probes E icon The Probes menu is displayed Probes EJ Raise Probes T Lower Probes Load Cartridge Unload Cartridge 1 Click the button of the action you need to take e Raise Probes e Lower Probes e Load Cartridge e Unload Cartridge Barcode Errors On the rare occasion that you receive a barcode error Wave will help you recover so that you can complete the run 47 Wave User s Guide Writing down the lot number serial number O2 values and pH values as soon as you open a Cartridge will save you time if you encounter a barcode error Consider including these details in the Notes section of the General Information window For details on how to enter notes see the General Information section Cartridge barcode read failure If there is a cartridge barcode read failure you will receive the following message Cartridge barcode read failure Unable to read cartridge barcode Click Rescan to retry Manual to enter the barcode manually or Cancel to cancel the Assay Rescan Manual Cancel It provides 3 options e Rescantoretry Click the Rescan button No other action is necessary e Manual to enter
22. 0 00 00 T Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 Measure 00 03 00 The following illustration shows an example of a Group summary GROUP Summary You can also add a summary of each of the ports 82 Groups Wave User s Guide Group Injection Pretreatment Media 7 Cell Type n mn B Bi n Ba 6 Ng N A no pyruvat no pyruvat pyruvate h pyruvate k pyruvate n no pyruvat N A N A No Pretreatmer No Pretreatmer No Pretreatmer No Pretreatmer No Pretreatmer No Pretreatmer N A N A No Medi No Medi No Medi No Medi No Medi No Medi N A N A No Cell Typ No Cell Typ No Cell Typ No Cell Typ No Cell Typ No Cell Typ N A Wave User s Guide To add the Port summary 2 Click the Port button The following illustration shows a part of a Port summary Port SURETY Volume 25 ul l Background Well assay media Concentration 0 uM Solvent pyruvate Concentration 10 mM Solvent Ke e e MM M M e Kee e e oM oM oM Kee SM M oM o Ke e e BE EP Hm M m Kee FE FE EP HM HM Hm NNN NNN N ON NNN NNN N ON W U U U UU UU Uu wwwwwwiw u NNN NNN N ON DNNNNNN WD B 1 1 1 1 1 1 B 3 pyruvte Concentration 10 mM Solvent 96 Friendly reminder Make sure you fill the ports of your Backgrour 83 Print Wave User s Guide It also has a print button that displays the following Print dialog box T Add Printer E Fax aah Adobe
23. 4 100 74 179 74 1215 55 1314 35 10 00 14 67 17 34 132 62 23 53 47 93 38 32 109 60 99 52 170 68 169 92 286 53 288 17 m X EE 14 67 1436 24 06 20 68 38 51 47 07 11041 104 81 175 79 194 47 281 65 294 91 a e a 113 04 700 2359 1764 37 23 34 18 11021 102 34 174 60 180 87 292 83 297 37 1377 1781 120 48 22 51 33 03 40 80 11178 102 83 189 92 186 98 267 98 295 53 a e AAA A 10 95 114 12 43 75 17 31 19 28 37 96 109 85 92 97 178 80 175 92 291 05 1275 12 1121 16 82 23 21 2126 29 12 135 56 1106 19 195 31 1167 89 1160 91 1278 95 1268 18 m NA 10 00 117 13 13106 1887 132 994 33 09 98 78 96 15 179 76 1160 70 1288 72 10 00 71 72 Wave User s Guide Wave User s Guide To eliminate outliers 1 Click on any well that is an outlier The background color of the well will change to gray to reflect that it is no longer part of the rate calculation on this tab To include a well that you previously eliminated 1 Click on the well again and Wave will include it in the rate calculation and its background color P6 will return to white To change the measurement point 1 Click on the downward pointing arrow to the right of the Measurement field and select the measurement point from the dropdown list i XFe Cell Mito E save x Add View n l OCR pmol min mum 3 Injection 1 10 00 Injecti
24. 5 SD 6 98 E ec 3 10k D PC 3 20K B coax Mean 36 52 SD 6 76 Mean 103 80 SD 6 10 Mean 178 91 5D 13 51 B pcs 80k Mean 287 24 SD 12 38 This window has two functions e tactsas the legend for both the kinetic graph and the rate details grid and bar chart The group colors shown in these visualizations correspond with the group names listed in the Group Details pane e tcan display group statistics To view the group statistics select the Details button in the upper right corner of the window which displays the mean and standard deviation for each group in the experiment If you prefer to see the standard error of the mean SEM you can change the display from standard deviation to SEM Read Standard Deviation and Standard Error of the Mean for how to change from one to the other Note Wells that you have previously excluded in the Rate Details grid will automatically be excluded from the calculation of these metrics Double clicking on the colored square for the group will prevent the group from being displayed on the kinetic graph but will not exclude the wells from these calculations However the mean and standard deviation of wells in excluded groups will change to Mean 0 00 and Standard Deviation 0 00 Double click the square again to turn a group back on Analysis OCR vs ECAR View The OCR vs ECAR view allows you to see a kinetic graph that displays the OCR on the Y1 axis and ECAR on the X axis To d
25. E Generate Groups Injections 6 Edit Pretreatment E Ww Pretreatments 1 Growth Medium F XF Assay Medium Pretreatments 7 Description DMEM 25mM Glucose 1mM Pyruvate 5 Fs ete Cell Type 4 Enterthe name of the pretreatment in the Name field and provide record keeping details in the Description field Assay Conditions XF Assay Medium The XF Assay Medium condition describes the assay medium used in this assay Record the Base medium and supplements for each medium used in your assay in these fields To define the medium 1 Touch or click the XF Assay Medium bar 2 Touch or click the Add Media Condition button 3 Click on Media 1 to display the Edit Assay Medium window 4 Enterthe name of the Assay Medium Condition in the Name field 5 Click on the Add button 21 Wave User s Guide 6 Click on More Details to display fields that provide record keeping details In the following example the assay medium is named XF Assay Medium lV nditions j j NC i Add X Remove Duplicate E E Generate Groups Injections s Edit Assay Medium Name gt V Pretreatments Te 4 amp XF Assay Medium 1 F Assay Medium s Add Remove X 8 Cell Type More Details Source Lot Number Prepared By Preparation Date 8 29 2013 Supplements pH 0 Assay Conditions Cell Type The Cell Type condition describes the source s of cellular material used for t
26. Group link to see Group information e Click on each of the port links to see Port information 36 Wave User s Guide General Information The General Information section provides information about the assay that you can edit from this window You can supply or edit any of the following fields e Project Information o Project Name o Principal Investigator o Project Number e Plate Information o Well Volume You cannot edit this field on a per assay basis See the Note below o Plated By o Plated On e Advanced Settings o Email Recipient List For more information see Email Recipient List o Advanced options For more information see Advanced Options e Notes Note Change the default well volume for your instrument from the Instruments tab under the Options section of XF Home For more information on changing the well volume see Instrument Tab Email Recipient List Under the Email Notification tab of Advanced Settings you can specify an email recipient list for this specific assay To add an email address 1 Enter an email address in the e mail address field under Email Recipient List 2 Press the Add button To remove an email address from the list 1 Select the address 2 Press the Delete button Advanced Options Under the Advanced tab of Advanced Settings you can change the port volumes and the buffer capacity for this assay and can check the software version and file version numbers 37 Wave User
27. Hew myacccuntgoutleok com KATE ini amp mtp liee cem Open MyParsword Port 3BJ Catalog ig Enable 55L lt Test Settings E Save kd Cancel In order to have Wave notify people of the status of every assay that you run 1 Supply an email address in the Mail From field and supply a password for this address in the Password field Specify an SMTP address in the SMTP Address field and the access port in the Port field Check Enable SSL if your IT configuration requires it Type the name of each recipient in the email address field under Recipients 4 5 Click the Add button 6 Repeat Steps 2 and 3 for each recipient When you have finished adding recipients press the Save button To check that your mail settings are valid 1 Click the Test Settings button after adding yourself to the Email recipients 2 Check your email to ensure that you received notification 3 CheckJunk E mail if you do not see it in your Inbox To take names off the list of recipients select the name and press the Remove button 59 Wave User s Guide Chapter 4 Analyzing Your Assay This chapter describes the 4 different views available to you and explains how to customize The 4 task oriented views that you have to aid in your analysis are as follows e Overview e OCR versus ECAR e Plate e Data View Summa Another feature of Wave is its ability to help you summarize your work using the Summary section For more in
28. Pr be FED cet Re pvp EB e Gi eir ee PEE Ie por tueid 64 Append to SUMINA seresa NEM Stet eso 65 To add all the information in the overview to the Summary document 65 To add only one item from the page such as the kinetic graph eeeesss 66 To see what you have appended in your summary document 66 Rate Detik and Bar Grap EE THUS MESE Rao E EUIS EIQUE Ee sUUT EENE 67 DE COMPONE 67 ELETE tA i PREIE EEA E T E T E E E N ET E EA E scant E A A A 68 Group Detalls RET m 69 Analysis OCR Vs ECAR ViCW cereias 69 Analysis Plate VIEW saasersaccanssndnsnisoainnssteteanesnasanrse tisunadeidauaes TES EXE ORDIN GUN eriadan arreire 71 FS IMM AUS 73 To include a well that you previously eliMiNated cccccecessssssececeececeuesececeeeeeseauanseeeees 73 To change the measurement point rore tror trt ri ero aet rra ao ee nen Vara EY e Ee aevo dre 73 MOPS lay the group details casas cate aisecetcs mm 73 Analysis The aA aia 74 4 Wave User s Guide T display the Data VIBWS siscvosbtastrasu nieee a TUER RAT Pubak NE EE EE EA R 74 Export to Excel from any View ccccccccccecsssesecececcecceaussseeceeceeseaeusaseeeceeeesesuaeaseseceeeesesaugaeseeeess 79 To export data from here or from any analysis view sesesseeececcececesssseseceeeeeesauaeeeeeeess 79 Summary Palos 80 EE e M
29. Pressthe Save button 45 Wave User s Guide Checking the Alarm On box will cause Wave to send you an email notification when the tray temperature exceeds the tolerance range In order to receive this notification you must specify your email address under Options For details on setting up your email address see the Advanced Tab section To turn off the alarm 1 Uncheck the Alarm On box 2 Press the Save button To reset the alarm after receiving notification that the tray temperature has exceeded the tolerance range 1 Check the Reset Alarm button 2 Press the Save button After resetting the alarm you can turn the heater on or off depending on the direction that the temperature exceeded the tolerance range by checking or unchecking the Heater On box y Heater On By checking the temperature before you start a run you can ensure that the tray temperature starts within the targeted range If the temperature exceeds the targeted range during an assay contact Seahorse Bioscience Support Tray Control Prior to a run after a run is complete or after a run has terminated due to an unexpected error it is sometimes necessary to eject or insert a tray If you need to eject or insert the tray 1 Click on the Tray icon The Tray menu is displayed 46 Wave User s Guide 2 Click on the Tray Out button to eject the tray or click on the Tray In button to insert the tray Probe Control Prior
30. Run W f i AKI p Remove Duplicate ue Senecate Groves tu FF Ze A Injection Condition 4 Injections a W eee Cell mito stress test Pretreatments Lg Background E amp 4 IE XF Assay Medium A B C D d Add 4 Cell Type b Compowsd rome ces uPC 3 LOK No Meta gt PC 3 2 5K PC 3 20K PC 3 40K PC 3 5K PC 3 80K To add a group 1 Click the Add button which is circled under the Well Groups window 2 Define the variables that make up the group by choosing from the dropdown list of conditions that you have defined 3 Repeat steps 1 and 2 to add another group After defining all of the groups you are ready to map the groups to the well plate 24 Wave User s Guide Map Groups to the Plate Map The Plate Map is where Wave displays information about each well in the experiment Wave uses group assignments to calculate group statistics in the Analysis file Groups There are 3 ways to determine the number of groups in your assay Use the Distribute Groups command assign them manually or add groups in the Plate Map tab Here we describe the shortest method using the Distribute Groups command in Wave Distribute Groups Wave can distribute groups across a plate if you push the Distribute Group command This command determines the maximum number of replicates and distributes the groups in vertical sections across the plate When the Well Groups list
31. Support web page Run Di ic T Diagnostic Test The Run Diagnostic Test button performs a test run on your XF instrument and sends the results to Seahorse Bioscience Support You will need to load a plate in order to perform this diagnostic and Support will provide you with instructions if they ask you to perform this test After running the test you can find the results in a tmp file under My Assays Version The current version of Wave installed on your computer or on your XF Analyzer XFe Home Instrument Modes In Wave Desktop under the heading NEW ASSAY there are no instrument tabs if you have a single type of analyzer an XF 24 or an XF 96 installed on your desktop and no Hypoxia Mode installed However if you elected to install more than one instrument type such as XF 24 and XF 96 or the hypoxia operating mode there is a tab for each instrument type and mode In Wave installed on your XF Analyzer under the heading NEW ASSAY there are no instrument tabs unless you have Hypoxia Mode installed If you do have Hypoxia Mode installed there is one tab for the instrument itself and one for running your assay in Hypoxia Mode Note If you would like to install Hypoxia Mode on your XF analyzer please contact Seahorse Bioscience Support 13 Wave User s Guide The first step in creating a design file or template if you have Hypoxia Mode or if you have more than one instrument type is to choose the instrument typ
32. ate Beg fees his lass Ns Masia Bb rate low im pyravate kri Kx tz 2 From this window you can change the group conditions and the plate map for an experiment that you have run This allows you to correct mistakes in the experiment design or fill in additional details 86 Wave User s Guide For instructions on how to define conditions and change the plate map refer to Defining Groups and Conditions and Mapping Groups to a Plate 3 When you are finished editing the appropriate fields touch or click the Apply NA Apply button located in the upper right hand corner of the window to make changes to the analysis file You can also cancel out of this window if you do not want your changes to take effect Click the Cancel a Cancel button to cancel your changes Assay Properties Assay Properties displays the Assay Properties window where you can modify and add information after running your assay To modify the assay properties 1 Click the Modify button and select Assay Properties from the dropdown menu Hill iti Hii Groups Conditions Normalization Your Wave software now displays the Assay Properties window 87 Wave User s Guide fi GS XFe Cell Mito Stress EJ A XFe Cell Mito Stress EJ Assay Properties GENERAL Information Project Information Plate Information Advanced Settings Project Fama n Pyruvate metabalim Pramil rair brapeect IKum
33. ate low fccp No Pretreatments No Media No Media No Media No Media N A N A N A pyruvate high fccp pyruvate low fccp No Pretreatments pyruvate medium fccp No Pretreatments no pyruvate high fccp2 No Pretreatments Wave User s Guide Port Summary The Port A Port B Port C and Port D links allow you to review the contents of each injection administered through a port along with the following details e Injection concentration e Name of the solvent if you specified one e Percentage of solvent used in the injection e Background wells e Port volume To change these injections click on the Group Definitions tab prior to running your design The following illustration shows an example of an injection port summary A Summary H ee E H i H re BE r E b b b E oH B Ha 2 E E Bio b ho Pho Plo B o P4 ds Pl Bd Ba LE Lt Pd FI Bl d Fi Fi FS FS hi he P Fi em uu ud a a Lu a 1 ud wa tao 4 o ko kg ww da a wu S Bo B B BB o ROM B B Bon M oR P M M EL l B 1 1 1 1 1 1 B Friendly reminder Male nire you W the pons of your Background Wells Print Summary Print Summary The Print Summary button prints a summary of your design on a printer or to a PDF file To print a summary of your design Print Summary 1 Click the button It displays the Print dialog box 40 Wave User s Guide General Select Printer ay Send To OneNote 2010 Snagit 11 Number of cop
34. ation and is recommended which is why it s checked Calibration Calibration tests ensure the accuracy of your instrument and are always the first step in a protocol therefore you cannot omit the Calibrate command Equilibration Equilibration ensures temperature stability before beginning your assay Measurement Cycles A measurement of the Oxygen Consumption Rate OCR and another measurement of the Extracellular Acidification Rate ECAR are included by default at various points during an assay 1 a basal measurement prior to the first injection predefined 2 ameasurement after each injection Injections are user defined for each port Seahorse recommends three measurement cycles for each metabolic rate A measurement cycle consists of 3 commands Mix Wait and Measure The Mix instruction directs how long the instrument is to raise and lower the sensor cartridge probes to ensure that analytes or compounds following injections are uniformly dispersed within the medium in each well The Wait instruction required for XF 24 only directs the instrument to delay a specified period of time after the Mix instruction before taking a measurement 26 Wave User s Guide The Measure instruction directs the instrument how long to record the flux of analytes in the transient microchamber once the sensor cartridge probes are lowered XF Wave software allows you to add additional measurement cycles as needed
35. ber ud 2 From here you can change or add to fields under each of the headings e Project Information e Plate Information e Advanced Settings e Notes For more information on editing assay properties refer to General Information 3 When you are finished editing the appropriate fields touch or click the Apply N ne button located in the upper right hand corner of the window to make changes to the analysis file You can also cancel out of this window if you do not want your changes to take effect Click the Cancel C button to cancel your changes Injections To change the names of your injections 1 Click the Modify button and select Injections from the dropdown menu 88 Wave User s Guide Groups Conditions eoee Assay Properties v Injections Normalization 2 From here you can change the name of each injection f t7 XFe Cell Mito Stress E3 Oc XFe Cell Mito Stress 3 Injections Change the name of your inje 3 When you are finished editing the appropriate fields touch or click the Apply NA Apply button located in the upper right hand corner of the window to make changes to the analysis file You can also cancel out of this window if you do not want your changes to take effect Click the Cancel CA Cancel button to cancel your changes Normalization Normalization is to scale data such that data from different microarrays can be compa red
36. bs in this Excel spreadsheet Assay Sorts Configuration Calibration Level Rate Rate Plates and Operation Log The following section describes how to design an assay in Wave 11 Wave User s Guide XF Home Page The first page that you see when you log into Wave is the home page The following illustration shows the purpose of most of the commands buttons and icons on the XF Home page Creates a new fi design file or If you have more than one type of analyzer such as an XFe24 and an XFe96 or an analyzer with Hypoxia Mode there is an Instruments bar If applicable select the analyzer or the analyzer with Hypoxia Mode n Feri Feld Hyposis bnde existing design Ro file or analysis file Saves assay conditions to Any templates that you have simplify design created or imported are shown in the Templates area Allows you t determine protocol Wave provides you with a defaults and other blank template to start your settings own design Provides contact Removes templates you no longer need information for customer support Moves a copy of a template and access to this to a different directory or guide storage device Imports additional templates XFe Home On the left hand side of the screen under XF Home there are 4 options for designing and managing your assays New Displays any existing template files and allows you to create a new template or as
37. e Cell Mito Stress EJ PU pue Rome summary Overview x Data view EJ Bo Medir Group Data Rate Level Data Raw Calibration Calibration View Event Log Background Cernectis Memurement Grouphame i li rie 1 ECAR Eror I Bachgeand Lone pynewhe los Icon b appret miiu LAEI 141 14527 i cx pgyrwvaie high fog ia 143 41507 E pma kw fij 147487 1441 286407 1 grusi mendum ier liri 143 20375 14013351 T 040 104 ACHT VOTES aono X 200030 r rus Peis mecum i nc pruvate high ccc TEM LLL HIS Birth it i Les 2 pynrayabe lice feep E5358 138 6145 5L2I3 S5 5ESES l pute metum te TESTS T 1000847 7 gueasabe hgh Geop TES35d MP nc gas d Eadhgeund LE 25 j 1531355 Y ee pynecste lcs Aon LA zT550 142 114 158 Reo You can find both the raw fluorescent data and the calibration data from this view To view a particular data set click on the specific tab within the window In the following example the Rate tab shows the rate data in a grid format 75 Wave User s Guide Group Data Rate Level Data Raw Calibration Calibration View Event Log Background Correction Measurement Well Group OCR A01 Background 0 81746 2 10242 0 00000 0 00000 A02 Group 2 0 81746 1 98904 0 00000 0 00000 A03 Group 2 0 81746 1 26549 0 00000 0 00000 A04 Group 2 0 81746 142193 0 00000 0 00000 Group 3 0 81746 0 71342 0 00000 0 00000 Group 3 0 81746 0 60547 0 00000 0 00000 Group 3 0 81746 0 22374 2 91399 5 18225 Group
38. e XFe Analyzer is not networked 1 Copyor move your analysis file asyr from the touch screen to a USB flash drive 2 Transfer the file from the USB to your desktop computer 3 Double click on the analysis file on your desktop computer to start analysis Note If the Wave software is already open on your desktop click the Open command from your XF Home screen and Browse to the location of your analysis file to open it For more information on analyzing your assay data see Analysis Overview Wave User s Guide Step 4 Transfer your assay data to a Microsoft Excel Spreadsheet The Wave software allows you to export XF data into a multi tab Excel spreadsheet Wave User s Guide To transfer your assay data to Excel for custom analysis 1 E 1 Pressthe downward pointing arrow on the Save button in the top left corner of your analysis screen 2 Select Save as to bring up the Save Assay dialog box ii XFe 2 0 0 40 87 10 16 A XX Smoke28 96Hypoxia EJ m Save x d 7 Summary Overview Ed E save S 1 idc OCR Wf Background Correction StdDev Display Group Yl Rate Y2 None Tes Options Smoke28_96Hypoxia Injection 2 T E E a cc J 10 Minutes Your Wave software will display the Save Assay window Wave User s Guide 3 Click on the Save as type dropdown list at the bottom of the window This list gives you 3 options Assay Analyze asyr Assay Design asyd and E
39. e by clicking on the instrument tab or to choose the instrument in Hypoxia Mode by clicking on the Hypoxia Mode tab XF 24 Design and run assays on 24 well plates XF 96 Design and run assays on 96 well plates XF 24 Hypoxia Mode Design and run assays in an XF 24 installed in a hypoxia chamber XF 96 Hypoxia Mode Design and run assays in an XF 96 installed in a hypoxia chamber Note If you do not see all of the instruments that are in your lab on the Desktop version of Wave you can install them yourself For more information about installation see the Installation Guide or contact Seahorse Bioscience Support Note The XF Analyzer touch screen will arrive with the appropriate configuration of Wave pre installed To add Hypoxia Mode please contact Seahorse Bioscience Support XFe Home Templates Any templates created or imported will appear in the Templates area beneath the Instruments tab s The example below shows the Blank template Ed that Wave provides to help you start your first design NEW ASSAY amp XFe24 XFe24 Extracellular Flux Analyzer XFe24 Hypoxia Mode XFe24 Analyzer installed in a Hypoxia Chambe For more information about templates see Templates Definitions for Templates Assay Design Files and Analysis Files View and manage files from the XF Home page The New command provides access to a Templates window that allows you to view and manage all of the templates that you have created 14
40. e displayed in the Y1 axis title on the kinetic graph when normalization is selected For instance if you are using cell count for normalization enter Cell into the Normalization Unit field and normalized OCR will be displayed on the kinetic graph as pMoles min Cell When you are finished editing the appropriate fields touch or click the Apply C Ariy button located in the upper right hand corner of the window to make changes to the analysis file You can Wave User s Guide also cancel out of this window if you do not want your changes to take effect Click the Cancel C button to cancel your changes To enter normalization values manually 1 Click the Modify button and select Normalization from the dropdown menu Groups Conditions HER Assay Properties Normalization 2 Enter values into the Normalization grid manually 3 Follow Steps 5 and 6 from the previous procedure Normalize When you have normalized your data the Normalize checkbox will appear in the Overview and the Data View You can toggle the data between normalized and not normalized by clicking the checkbox on and off 91 Wave User s Guide Analysis with Excel To transfer the entire results file asyr to Excel for further analysis 1 Press the downward pointing arrow on the Save button in the top left corner of your screen 2 Select Save as to bring up the Save Assay dialog box Xm og aye A 2 Orga
41. ergvej 3 2100 Copenhagen Denmark 45 31 36 98 78 Seahorse Bioscience Asia199 Guo Shou Jing RoadSuite 207Pudong Shanghai 201203 CN 0086 21 33901768 Seahorse Bioscience qoo SU
42. es Documents v Search Documents p New folder Downloads S Recent Places EE Desktop Libraries ES Documents 2 Music s My Assays Pictures BE Videos B Mary Horvath iE Computer amp L Local Disk C eS DVD RW Drive 43 BD ROM Drive amp thermogenic x9 apps thmsrd Hide Folders Documents library Includes 2 locations Name XFe14 User s Guide L Timesheet Barcode My Assays XFelnstrument SpecificationAndWorkflow XFe Assays Sprints a Wave Design Team Meetings JJ XFe 1 1 1 Release Notes and ReadMe History of XF and XFe Template di Status Messages i Cell Seedina Arrange by Folder v Date modified 8 8 2013 4 28 PM 8 8 2013 10 00 AM 8 7 2013 9 58 AM 8 5 2013 12 52 PM 8 2 2013 12 38 PM 7 26 2013 12 31 PM 7 25 2013 2 52 PM 7 25 2013 10 12 AM 7 24 2013 1 48 PM 7 19 2013 3 28 PM 7 19 2013 12 13 PM 7 9 2013 3 54 PM 7 9 2013 3 50 PM 2 Select a directory for your graph data 3 Type a file name in the File name field 4 Save your data by pressing the Save button 94 Type File folder 7 File folder File folder File folder File folder File folder File folder File folder File folder File folder File folder File folder File folder b Wave User s Guide Seahorse Bioscience Inc 16 Esquire RoadNorth Billerica MA 01862 USA 1 978 671 1600 Lm i Seahorse Bioscience Europe Symbion Science Park Fruebj
43. est Modified 8 30 2013 Author mhorvath Description An example template Project Name Pyruvate metabolism Project Number 01 Investigator J Irons Template Details lists the following information Name the name of the design template Modified the last time you modified the template Author the author of the template Description a description of the template Project Name the name of the project if one was given Project Number the project number if one was given Investigator the name of the investigator if one was given Assay Design Files A design file asyd contains all of the information needed to run an experiment including groups and condition definitions plate map configuration and protocol definitions When you run a design file the XF Analyzer converts it from a design file asyd to an assay results file asyr If you plan to reuse an assay design file you must save it as a template For more information see Save and Save as Template 17 Wave User s Guide AN Analysis Files An analysis file asyr contains data from a completed run including all of the raw data and calculations as well as information about the experiment s design These files can either be used to analyze the assay data or saved as design files to start a new design Start a New Assay Design To begin a new assay design take the following steps 1 If you have an Instruments bar select the type
44. ew by clicking on this y button Analysis Overview When you open a result file asyr for the first time you will see the Overview which is the default view The Overview allows for flexible data analysis within Wave This window is where Wave displays kinetic graphs for all rates and where you can perform common tasks such as excluding outlier wells Group statistics are also calculated and displayed within this view To display the Overview if you have closed it 1 Select the Add View button in the top left region of the screen and choose Overview from the dropdown menu 61 Wave User s Guide l XFe 2 0 0 40 87_10_16 Display Group OCR VS ECA Plate Data View You can add any number of Overviews by selecting the Add View button in the top left region of the screen and choosing Overview from the dropdown menu This section describes the different parts of the Overview screen and explains how to customize each part to meet the requirements of your experiment e Kinetic graph e Rate details e Bar graph Kinetic Graph A kinetic graph is the most common way to display data from the XF Analyzer Display Group Y2 None XFe Cell Mito Stress Test Injection 2 Injection 3 E E ce ud kd Minutes 62 Wave User s Guide OCR ECAR or PPR There are three rates calculated by Wave using the XF Analyzer data collected during an assay e O consumption rate data OCR
45. formation on this feature refer to the Summary Tab section and look for the Summary section at the end of the description of the overview Arrows for Expanding a Graph or Chart The Expand arrow allows you to expand a graph or chart by clicking it In the following illustration the expand arrows are circled in red P XX XFe Cell Mito Stress EJ 4 toe oc Ada Vw Summary Overview EJ mem 1 m OCR wf Background Correction StdOev gt E oy Baseline OCA ipmol rren YE Rete Y Nore 9o Cptecs If you click the arrow that is circled in the middle of the screen the kinetic graph will expand horizontally as shown in the following illustration 60 Wave User s Guide Summary Chery x p Oc f Background Connection rner Format Sidbew EJ Cw Baseline 9 If you would like to decrease the size of the graph click the arrow that is circled on the right hand side of the graph Zoom Pan and Restore Buttons There are 3 buttons available on each graph and chart in the Overview and the OCR versus ECAR view The Zoom button allows you to magnify a particular part of the graph or chart by clicking this button and then dragging on the section you would like to magnify The Pan button allows you to move around the magnified area by clicking on this button and then moving the cursor around the graph or chart The Restore button allows you to return the graph or chart to its full vi
46. hat the information is complete Access to Predefined Conditions You can define conditions in the Catalog then when you start a new assay design these predefined conditions will be available for selection from the dropdown list for each condition You can define Compounds used in Injection Strategies Pretreatments Media and Cell Types f 7 New Design El tt New Xfe24 Assay Ed c tmp 01Bitmp Ed 53 Wave User s Guide To add a specific condition Choose the condition such as Compounds Fill out the appropriate fields describing this condition The Name field is required the other fields are optional 3 Press Enter Add the next condition by following steps 1 2 and 3 over again 5 When you are finished entering your condition definitions press the Save button on the top right hand corner of the page You can manage the conditions available on Wave by adding and removing them from the list To find the location of your catalog directory use Options Read the section Customizable Default Settings for more information You can also share these definitions by copying the catalog files to a USB flash drive and then pasting them into the Catalog directory of a different computer or a different XF instrument Customizable Default Settings The Options screen contains default settings on 3 different tabs General Instrument and Advanced M jnstrumeot Apps Advanced General Tab The General tab allows you t
47. he assay with information about the cell line seeding concentration and passage number To define the cell type 1 Touch or click the Cell Type bar then the Add Cell Type Condition button 2 Select Cell Type 1 to display the Cell Type window 22 Remove E Duplicate the Generate Groups Edit Cell Type Injections G e a4 Cal Ure E Pretreatments 1 Growth Mediuma Name Islets of Langerhsans ssay Medi F XF Assay Medium in p 1 Park i nth glucolu Lab a D Cell Type 4 anxreatc slet cells with glucokinase mutabon AAA XX Prepared By M Smith Passage Seeding Density 2000 Source J Godine MGH Prepared On 29 2013 Thawed 8 2 2 eMe 3 Enteradditional details in the appropriate fields Automatically Generate Groups Wave User s Guide Wave automatically generates groups based on the number of independent variables you define in your assay If you define only one specific condition Wave will assume that it is a global condition and will not use it to differentiate groups If you define more than one condition the software will calculate the number of groups assuming every possible combination of independent conditions Once you have finished defining all assay conditions touch or click the Generate Groups button x Remove Duplicate Gi Generate Georugs 7 9 Injections Edit Cell Type Tell mito ores test AE Cellline x Pretreatments Name 4 E XF Assay Medium Cell Type Seeding Dens
48. ies E Selection f Curent Page f Pages Collate 2 Selectthe printer Use the scroll bar circled in red to find the printers that are on your network 3 Select the Number of copies 4 Pressthe Print button 41 Wave User s Guide The following illustration shows an example of a part of a summary that was printed to a PDF file Seahorse Bioscience Analyze summary report Assay Name xFae Cell Mito Stress Test Project Name pyruvate metabolism Project Number 01 Principal Investigator J Irons Run Date 3 7 2013 6 19 AM PROTOCOL SUMMARY TOTAL TIME 01 0600 Initialization Basal Injection 1 Injection 2 E Calibrate LY Mix 00 03 00 Inject Part A Inject Port B tat Equilibrate 00 00 00 Wait 00 0000 Jr Mic 00 03 00 f Mow 00 03 00 Measure 00 03 00 Wait 0000 00 lt Mise 00 03 00 Measure 00 02 00 I Measures 00 03 00 Wait 00 000 Jf Mic 00 03 00 f Mi 00 03 00 P Measure 00 03 00 Wait 0000 00 Wait 000000 Lf Mix 00 03 00 l Measure 00 03 00 Measure 00 03 00 Wat 00 0000 E Measure 00 02 00 Injection 3 Injection 4 I Wait 00 00 00 o Inject Port C Inject Port D f Mix 00 03 00 JT Mix 00 03 00 Wait 00 00 00 Wait 00 00 00 P Measure 00 02 00 E Measure 00 03 00 f Mix 00 02 00 IT Mix 00 02 00 Z Wat 0000 00 Z Wait 00 00 00 P Meacure 00 0200 P Measure 00 03 00 Wave User s Guide Run Your Assay Once you have moved your design or template file to
49. ighlight v Show Injections Markers In this pop up window you may configure specific settings for each axis e The Maximum Minimum and Interval fields allow you to set the scale of each axis e The Thickness field allows for visualization of lines on the graph 1 thinnest 5 thickest that delineate each interval e The Show Error Bars box allows you to toggle error bars on or off See Standard Deviation and Standard Error of the Mean for more information e The Show Zero Line box allows you to display a horizontal line originating at the zero point on the x axis for reference e The Point to Point check box allows you to show point to point rates for a particular axis e The Show Rate Highlight check box highlights the selected measurement with a blue vertical marker e The Show Injections Marker check box shows when each injection occurred Append to Summary You can append all or parts of the contents of the overview to your summary in two ways To add all the information in the overview to the Summary document Ks l 77 J button in the top left hand corner of the overview screen 1 Click the Summary This will append all of the graphs and charts on the screen onto your summary document 65 Wave User s Guide To add only one item from the page such as the kinetic graph 1 Right click the graph and select Append to Summary from the dropdown list foomOut Copy Export Export Graph Data Print
50. ing the up and down arrows Protocol Defaults Cycles Mix Wait Measure 03 00 02 00 03 00 W Measure After Injection By default your instrument will measure after each injection If you do not want the instrument to measure after each injection you can uncheck Measure After Injection After making changes click the Save button at the bottom of the screen 57 Wave User s Guide Ports and Wells Seahorse Bioscience recommends that you use the default settings for Port and Well volumes however you can change the default port volume and the default well volume for each instrument Ports and Wells Port Volume Well Volume u 999 To change the port volume or the well volume 1 Type in the new value in the Port Volume field or the Well Volume field or use the up and down arrows to increase or lower the value to the correct amount 2 After making changes click the Save button at the bottom of the screen Advanced Tab From the Advanced tab you can specify people who will receive email notification at the following times e after calibration e after successfully finishing the run This email notification includes the results file as an attachment Wave also sends out notification if an error occurs during the run 58 Wave User s Guide The example below shows the configuration for sending notification through Outlook or Hotmail A e New Design x XF Home General instrument Advanced F
51. is complete touch or click on the Distribute Groups button Background Wells Wave will automatically default to Seahorse recommended background wells You can change the number and locations of these wells by clicking on the Background tab in the Well Groups window then double clicking or double tapping on the desired well to assign it as a background well ter vue 33 Ourente Joust i Ps 4 3 5 amp Q eo fe Ne Cat yon Define a Protocol After you have defined groups and conditions and mapped these groups to a plate you can define the assay protocol by clicking on the Instrument Protocol tab Here you will define the assay protocol including measurement and injection cycles Default Commands and User Specified Injection Strategies The predefined assay protocol includes the following steps 1 Calibrating 2 Equilibrating changeable 25 Wave User s Guide 3 Defining the Basal Measurement Cycle editable Note Specifying injections is entirely up to you There are no defaults However if you specify an injection you will usually take a measurement after the injection You can use a predefined measurement cycle for this purpose D ssa jix 3 Measurement Cycles Mix Wait Meas 03 00 02 00 03 00 v2 Calibrate The XFe always performs calibration to make sure measurments are accurate w Edit Measurement Details tw Equilibrate Equilibration occurs after Calibr
52. isplay the OCR vs ECAR view 1 Select the Add View button in the top left region of the screen and choose OCR vs ECAR from the dropdown menu 69 70 XX XFe Cell Mito Stress OCR vs ECAR Plate Data View Be eem E ce a con E B n wf Background Correction DE e XFe96 FCCP optimization plus minus obligo c 1 oE fae Tro f Wave User s Guide aan an io fase gt T i2 40 f t Tt A 7302 1053s fi Wave User s Guide Analysis Plate View The Plate view shows two types of information e Grid of the individual rate data points for each well at a specific measurement point e Group details that include the mean and standard deviation for the selected rate From here you can search for outliers so that you can eliminate them from the calculations An outlier is an observation that is numerically distant from the rest of the data Note Outliers eliminated in this view will not apply to other views You must manually omit them in each additional tab To display the plate view 1 Select the Add View button in the top left region of the screen and choose Plate from the dropdown menu Measurement Overview Display Grou OCR vs ECAR Plate y Data View E swe dmm Summary Overview E3 Plate EJ Measurement i m OCR Ww Background Correction EA Baseline OCR pmol mn 4 6 j a 10 10 00 9 64 29 02 26 73 39 38 39 96 109 2
53. ity G atisy medium PBC 3 PC 3 2 5 4 2 Cell Type ron et c 5 10 Prepared On Select a Gate Thawed Select a date 23 WELL GROUPS Ten a Background Duplicate Call mato erete tert zx assay medem B rcas o Cell mao stress test AF avery medn E cio Cali mato tree tet E F assay mediom pC 3 20K Ceti mato stress test FS WF assay medion B co Celi mito tress tent Z P assay mecum BB pc 3 80K B W Na gt Q c 32 y No Pretreatments 3t a PC SK W No Dretrestments oe PC 3 10K x No Pretreatments nr 5 sy PCJ 20K Wave User s Guide In this example 6 cell types were defined PC 3 2 5K PC 3 10K PC 3 20k PC 3 40K PC d 80K PC 3 5K while only one Injection Strategy and one XF Assay Medium were defined If you click the Generate Groups button Wave generates 6 groups generating every possible combination of Injection XF Assay Medium and Cell Type The combinatorial logic is 6 x 1 x 1 resulting in the generation of 6 groups If there were 3 Injection Strategies 1 XF Assay Medium and 6 Cell Types the combinatorial logic would be 3 x 1 x 6 generating 18 groups Manually Generate Groups Generate groups manually to select the conditions that distinguish one group from another yourself PR oS xFe Cell Mito Stress EJ 77 Cell density optimiz E3 E sre Group Definitions instrument Protocol Review and
54. lay OCR in the Rate Details grid In the previous illustration Injection 11 is selected therefore this measurement is highlighted in blue on the kinetic graph and the OCR rate measurements are reflected in the Rate Details graph You can exclude individual wells from the Rate Details grid so that the software excludes them from the kinetic graph and the group statistics data Touch or click on a well to exclude it excluded wells will show up as shaded wells with a black border When you exclude a well the software will not show it on the graph and will exclude the well from group statistics calculations Use the column numbers and row letters buttons to exclude entire sections of the grid Bar Graph You can also see these rates in the Details bar chart found below the Rate Details grid Here you can display the rate data either by group or by well Display Group c ame 2 3 2 68 Wave User s Guide To see the rate data by well use the Display dropdown menu circled in red on the preceding illustration and select Well You can use this tool to view group differences for a particular measurement group mode or to help identify outliers within specific groups well mode Group Details The final display found in the Overview tab is the Group Details pane which is on the lower left side of the screen Group Details gg Background gB oco B oss Mean 0 00 SD 0 00 Mean 13 04 SD 3 52 Mean 24 8
55. le from any directory to which you have access To import a template file select the Import button 15 16 The Import Assay dialog is displayed It allows you to browse through your folder directory to select an assay file Import Assa po y C C m My Documents My Assays Wave User s Guide Organize v New folder Favorites EE Desktop Jg Downloads El Recent Places EE Desktop 5g Libraries Documents RU Music tj My Assays i Pictures BE Videos B Mary Horvath di AppData i Contacts Fe File name Arrange by Folder v My Assays Name No items match your search m v XFe Assay Template asyt z The Export button allows you to export a template file for any of the following reasons To transfer it from your desktop to the XF Analyzer if it is networked O To transfer it to a flash drive if it is not networked o To transfer it to a shared directory to give your colleagues access to it o To back it up To export a template select the template and click or touch Export The Remove button allows you to delete a template file To delete a template select the template and click or touch Remove Wave User s Guide Notice Template Details The Template Details pane is to the right of the Templates pane The example below shows the template details for the XF Cell Mito Stress Test template Template Details Name XFe Cell Mito Stress T
56. lick on this B Background vertical bar to start B ou defining D Ne lncchans W No Preteratnents conditions S No Media v Ie No Cell Types B c2 o Ha irgectian T Ma Fretumatements 2E Hn Mecha aft Ha Cell Types Conditions Describe the 1 Group 3 Plate Map ronds for ch riim ILE o Ho irgechans x Ha Pretrmateriemls tn your Groups Map your Groups to che Piste He heda T we Ho Cell Types B Group o H irgectians T Na Prevesirents 22 Ne Mecha aft Ha Cell Types Assay Conditions Injections Injections describe the contents of each set of injections in the assay Each specific injection strategy represents the contents of up to 4 injections one for each of the 4 ports A B C or D To define an injection strategy 1 Touch or click the Injections bar 2 Touch or click the Add Injection Strategy F Add Injection Strategy button 19 Wave User s Guide 3 Touch or click on the name of an injection strategy on the left default is Inj Strategy 1 to display a dialog box where you can fill in the fields to name the strategy and describe the contents of each injection port In the example below 6 injection strategies were defined and they were named no pyruvate low fccp no pyruvate medium fccp pyruvate high fccp and so on db CDU xFe Cell Mito Stress EJ pe Group Definibons Instrument Protocol Review and Run This counter lets you know how many injection conditions zx Remove
57. nize New folder a Hr Favorites Documents library EE Desktop XFe Assays H Downloads 3 5 Recent Places Arrange by Folder Name a Cell density optimization with cell mito stress test a GlycolysisAndStressTests EE Desktop Libraries mito glyco E Documents a Music ix My Assays Pictures B Videos E File name XFe Cell Mito Stress Test Save as type Assay 4 Hide Folders 3 Navigate to where you would like to save your Excel file Type the file name in the File name field 5 Click on the Save as type dropdown list at the bottom of the dialog box This list gives you 3 options Assay Analyze Assay Design and Excel 2007 2010 xlsx 6 Select Excel 2007 2010 xlsx Click the Save button in the bottom right hand corner of the dialog box pt 92 Wave User s Guide To transfer data from a graph or chart to Excel for further analysis 1 Right click on the graph and select Export to Graph Data from the dropdown list QU XFe Cell Mito Stress E3 x XFe Cell Mito Stress EJ B Save Es AddView Summary Overview Ed Rate OCR W Background Correction PAE StdDe Y1 Rate hi Y2 None Display Group XFe Cell Mito Stress Test a E a EL o Export Graph Data Print Minutes Append To Summary This will bring up the Export to Excel window 93 Organize v i Export to Excel 3 Librari
58. ntthe summary e Export the summary to Microsoft Word Adobe PDF HTML or Rich Text The Summary tab displays a version of the protocol summary that you can edit and print P XX Xe Cell Mito Stress EJ t b A Vew Summary Overview EJ eias I 12 two Cie ee WE b U Parte 4 u eh x a a 2 ES aa gt Ma Cigiscant _ Font a Perpgeagh Ij Poges Tates Pictures Seahorse Bioscience Assay Name XFe Cell Mito Stress Test Project Name Pyruvate metabolism Project Number 01 Principal Investigator J Irons Run Date 8 7 2013 6 19 AM Notes This window provides a multi faceted tool bar SS XFe Cell Mito Stress 3 uii C uon 3 QU fi 7 YT information a PrintBunon T hs H eo Add View Summary Overview EJ Dataview Ej s S Me ie d Fi LS P 4 Verdana i a ae fi 4 kna picard Font iari Panait fa Lf This bar allows you to edit your summary with its editing tools e Cut copy and paste to the clipboard e Change the font its size color and style e Subscript or superscript a character or characters e Highlight some text e Inserta table a picture or a page break 80 Wave User s Guide Append information from your assay You can add the General information recorded as part of the assay design which is from the Review and Run screen of the design file To add General information 1 Click the General button from the Append section of
59. o Backoroung cmpd A 6i Boso e VVave XF eio E n 2 a M g Ha h FT mL E I BEEEEESEEEEE oro za awy Bus H cmpd B 6i cmpd 4 20 Wave User s Guide Contents Chapter 1 Designing Your Assa a cecsiveceicsineventesecesisixecoriieveeneeet 6 Overview of the Process Design Run and Analyze eee ee eee eee eee eene nennen enhn nno 6 Step 12 DESIGN your ASSAY PM m 6 SD2 iU TR TUM Lic e 6 To run a design created on the Wave software of the XF Analyzer 6 To run a design created on your desktop if the XF Analyzer is networked 6 To run a design created on your desktop if the XF Analyzer is not networked 7 Step 3 Analyze your assay dabas esee eir ben Eoo eerie Eve utu Ea ab eue HY ERE ee vEbTY Ee ruv DE x Ee EEY Pedo PUR V MER EE dins 7 To transfer your analysis file to your desktop if the XF Analyzer is networked 7 To transfer your analysis file to your desktop if the XF Analyzer is not networked 7 Step 4 Transfer your assay data to a Microsoft Excel Spreadsheet eeeeesesss 8 To transfer your assay data to Excel for custom analysis ceeececcceeeesecceeeeececesaueeeeeeteas 9 ME Home Page 12 XF TOANE NIANON AEOS 12 PN rains EOT ean AEA AE E T E EET AE A A ETE 12 S EEE E esc cet ci A A A AE EAEE ee A E EE ea E E 12 COE a 6 12 Op
60. o change your password or user name under Login Settings It also shows the default settings for buffer capacity and atmospheric pressure 54 Wave User s Guide Default Capacity 0 00078 mol L Atmospheric Pressure Default Value 760 0 mmHg Buffer Capacity Seahorse Bioscience determined the default buffer capacity setting for XF Assay Medium You can change the buffer capacity by typing it in the Default Capacity field The Wave software uses the buffer capacity to translate ECAR Extracellular Acidification Rate readings to PPR Proton Production Rate You can also change the buffer capacity in the assay design file see Reviewing and Running an Assay Advanced Settings Note Buffer capacity changes based on the constituents of the medium different constituents have different capacity to buffer the medium from pH changes Atmospheric Pressure You can change the atmospheric pressure if you are running an assay at high altitudes or if the atmospheric pressure in your lab differs from the default value of 760 0 mmHg These are settings that you can change from one assay to the next but that are usually defaulted to specific values Recent Lists With Recent Lists you can change the maximum number of directories and files that Wave displays under My Places and My Assays by typing in the new values or using the up and down arrows Number of Recent Places Number of Analysis Files Number of Design Files
61. ocation of your design file to open it Wave User s Guide 4 Touch or click the Review and Run tab of your touch screen 5 Click the START RUN button To run a design created on your desktop if the XFe Analyzer is not networked 1 Save the design file to a USB flash drive 2 Plug the flash drive into the USB port below the touch screen of the XF Analyzer 3 Copy your design file asyd to the local file system 4 Log onto the Wave software on your XF Analyzer s touch screen by double tapping or fr double clicking the shortcut Bir 5 Double tap or double click on the design file on the screen of your XF Analyzer If Wave is already open on the touch screen click the Open command from your XF Home screen and Browse to the location of your design file to open it 6 Touch or click the Review and Run tab on your touch screen 7 Click the START RUN button For details on reviewing and running an assay see Review the Design of Your Assay and Run Your Assay Step 3 Analyze your assay data You can perform data analysis on the touch screen however Seahorse recommends analyzing results files asyr on your desktop for maximum functionality To transfer your analysis file to your desktop if the XFe Analyzer is networked 1 Move or copy your analysis file asyr to your desktop computer 2 Double click the analysis file on your desktop computer to start analysis To transfer your analysis file to your desktop if th
62. of instrument or the type of instrument with Hypoxia mode If you have only one instrument with no Hypoxia chamber you will have no Instruments bar and do not need to select an instrument or mode In this example to run your assay on an XF 24 with Hypoxia Mode you would select the XF 24 Hypoxia Mode tab E XFe24 XFe24 Extracellular Flux Analyzer XFe24 Hypoxia Mode k 2 Select the Blank L5 template from the Templates window a 3 Click the Design Design button This will create a new Design ee New Design E3 tab where you can begin to design your assay Define groups and conditions See Define Groups and Conditions Map groups to the plate See Map Groups to the Plate Map Define the assay protocol See Define a Protocol Review and run your assay See Review and Run oS T X Open the analysis file asyr to review and analyze assay results data See Analysis Define Groups and Conditions The Group Definitions tab is where you can define Assay Conditions Well Groups and Plate Maps You can add groups manually or define the assay conditions and have Wave generate groups automatically The 4 different types of assay conditions that you can define are listed below e Injections e Pretreatments e XF Assay Medium e Cell Types 18 Wave User s Guide To start to define conditions click on the vertical bar of the Conditions panel on the left side of the screen WELL GROUPS Te C
63. on 2 To display the group details 1 Click on the Details button of the Group Details column on the right side of the screen Group details will change as you eliminate outliers 73 Wave User s Guide Group Details E Background Mean 0 00 SD 0 00 ER no pyruvate low fccp Mean 133 27 SD 741 BE no pyruvate medium Mean 140 76 SD 8 57 ej pyruvate high fccp Mean 133 74 SD 7 10 Es pyruvate low fccp Mean 143 88 SID 5 02 E pyruvate medium fc Mean 143 82 5D amp 38 E no pyruvate high fcc Mean 144 73 SD 7 33 Group Details shows the mean and standard deviation for the group rate If you would like to change standard deviation to standard error of the mean refer to the Standard Deviation and Standard Error of the Mean section Analysis The Data View The Data View provides many different non graphical views of the data and the run including the following e Raw data level data group data and well data e Rates OCR ECAR and PPR e Calibration for each well and group e Aneventlog of the run Each tab in the data view is exportable to an Excel file To display the Data View 1 Click on the Add View button and choose Data View 74 Wave User s Guide Measurement Display Group OCR VS ECAR Plate The Wave software brings up the Data View which shows the raw data in three main forms group data rate data and level data lt lt XF
64. ou can also elect to view the O or pH level data or rate data for each well rather than the group To view data for each well on the graph select Well from the Display drop down menu Standard Deviation and Standard Error of the Mean You can specify whether you would like to see the standard deviation Std Dev or the standard error of the mean SEM by choosing from the Error Format dropdown list B Save v mfa Add View Summary Overview Eg Measurement 1 AA OCR W Background Correction SAALE StdDev Display Group Wie Rate Y2 None XFe Cell Mito Stress Test If the error bars do not display check to make sure that you have enabled them See Kinetic Graph Customization for more information on enabling error bars Kinetic Graph Customization Ry Options You can manipulate the kinetic graph visualization by selecting the Options button located in the upper right hand corner of the pane 64 Wave User s Guide Kinetic Line Chart Options Graph Title yee Cell Mito Stress Test Y1 Axis Max a tTJU U wv Auto Scale Interval Thickness a L JC v v V Show Error Bars vd Show Zero Line f Line Markers Point to Point Y2 Axis Min Max a A v v d Auto Scale Interval Thickness h L v v v d Show Error Bars Show Zero Line Line Markers Point to Point Time Axis Min Max a Rm alk Interval Thickness a h JC v v H
65. ou can save the file in either of two formats as a design file or as a template Note Select Save As Template if you plan to re run this assay or expect to make small changes to it and then re run it To save your design as a design file 1 Click the downward pointing arrow on the Save option to save it as a design file asyd QE cE NewD LC save Group ias m Save As l Save As Template The Save Assay browser is displayed 29 button and choose the Save As 2 8 My Documents XFe Assays Organize New folder i Mam Yr Favorites E Name EE Desktop _ XFe Cell Mito Stress Test asyd a Downloads el Recent Places BE Desktop Libraries ES Documents a Music ial My Assays Pictures B Videos 4 File name Mew Xfe24 Assay Wave User s Guide Date modified Type 8 14 2013 5 53 PM ASYD File T Hide Folders 2 Enter a name for your new assay in the File name field 3 Select the Assay Design type from the Save as type dropdown list 4 Click the Save button to save your design file Use this option if the assay is not one that you are planning to repeat Once the instrument has completed the protocol Wave generates a results file for data analysis To save your design as a template 1 Choose the Save as Template option l Save As Template The Save As Template dialog box is displayed 30 pr dee de 9 31 Wave User
66. r template file from the Import Assay window Import Assay QU LocalDisk C ProgramData Seahorse Bioscience Inc Seahorse Wave Templates Search Templates pP Organize v New folder e e E My Pictures gt Name Date modified Type Size BE My Videos m Saved Games J Searches 9 SyncFolder jE Computer L Local Disk C d downloads Drivers 4 Firefox J Hotfix B MSOCache d PerfLogs d Program Files d ProgramData d Users 4 D IE 2 Cell density optimization asyt 8 29 2013 11 18 AM ASYT File _ Hypoxia Assay FXe96 asyt 8 6 2013 5 47 PM ASYT File _ Hypoxia Assay on XFe96 asyt 7 31 2013 4 33PM ASYT File m Filename Cell density optimization asyt v XFe Assay Template asyt v Open Show previous versions Click on the Open button and choose the Open option from the dropdown menu 5 Click or tap once on the template icon and choose the Design button See an example of a template icon below 6 Make any changes you need to make prior to saving the template as a design file 7 Click on the Save 8 Type the name of your design in the File name field button in the top left corner of the window 9 Select Assay Design in the Save as type field 10 Click on the Review and Run tab Review the Assay Design From the Review and Run window you can review your design and run the assay From here you can
67. ries that you access through Wave but are not defaults are shown in grey scale ayp Recent Assays Click on Recent Assays to access your most recent analyses and designs under the My Assays pane regardless of their location 4b My Assy O ER Click on My Assays to access the analyses and design files under your My Assays directory Click on Temp to access the tmp file created if you ran the diagnostic test on your XF instrument This test is found in the Support pane that you can access on the Help page Click on Templates to access any assays or analyses that are stored under your Templates directory You can find the most recent modification date device name project name project number and investigator s name by hovering over an analysis file or a design file Set the maximum number of files listed by selecting Recent Places in Options Similarly you can also set the maximum number of files listed under Analyses and Designs in Options For more information on Options read Customizable Default Settings Bl rose The Browse button at the bottom of the My Places pane allows you to navigate to additional assay design files and assay analysis files You can open current assay files asyr Wave 1 0 1 1 files asy and XF files xfd from the Open Assay window that is displayed when you click the Browse button Note Although Wave software will convert XF data from previous versions check the converted files to ensure t
68. rument Tab The Instrument tab shows the instrument or instruments that are available in your lab and allows you to set the default values for protocols port volumes and well volumes There are 4 possible instruments that your lab might have 56 Wave User s Guide The XF 24 instrument allows you to perform your assays on 24 well plates The XF 24 instrument with Hypoxia Mode allows you to perform your assays in a hypoxia chamber The XF 96 instrument allows you to perform your assays on 96 well plates The XF 96 instrument with Hypoxia Mode allows you to perform your assays in a hypoxia chamber XFe96 Hypoxia Mode Select the type of instrument where you would like to perform your assay then select the other default values for this instrument while you are on the Instrument window From here you can enter default values for e the number of cycles in a protocol e the number of minutes and seconds for Mix Wait and Measure e the port volume e the well volume The next sections describe how to enter these values Protocol Defaults Seahorse Bioscience recommends that you use the default settings for Port and Well volumes however in some cases it may be necessary to change these settings You can change the default times for mix wait and measure and change the default number of cycles by typing in the new values or by us
69. s Guide Advanced Settings Email Notification Advanced Port Volumes A 4 A ull B ul A A a m e b e a To change the port volume for this assay 1 Type in the new value in the Port Volume field or use the up and down arrows to increase or lower the value to the correct amount To change buffer capacity for this assay 1 Scroll down to the Buffer Capacity section under the Advanced tab Advanced Settings C 25 u D 25 Ju viv Buffer Capacity Capacity 0 00078 mol L 2 Enter the desired capacity in the Capacity field To check the version number of the software and of the design file 1 Scroll down further You will see the version number of the Wave software in addition to the version number of your design file under Version Information Protocol Summary The Protocol link allows you to review the protocol that you have defined for your assay To change the protocol click on the Instrument Protocol tab to edit the protocol prior to running your design refer to Define a Protocol The following illustration shows an example of a protocol summary that the Protocol link could display 38 Summary PROTOCOL SUMMARY Protocol Group Initialization Port A Calibrate isid ili Equilibrate 00 00 00 Port C Port D Injection 3 Inject Port C YW Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 JT Mix 00 03 00 Wait 00 00 00 I Measure 00 03 00 Group
70. s Guide Save As Template Enter the name of your assay template in the Name field required Enter your name in the Author field optional Enter a description of your assay in the Description field optional Click the Save button Choosing to save your design files as templates allows you to open the template as a starting point for a design file You are able to create a new design file repeatedly making small adjustments to the fields as needed thus saving time Wave User s Guide Template files are stored under the Templates directory which you can find in the My Places section of XF Home The following example shows how you can locate your template files from within Wave Woo DTU MM LL BEEN 3 XFe 200 4 CUZ New Design EJ My Places My Assays Recent Assays e ae Analyses Catalog db Hel mito glyco Options P File b Help a My Assays esi Cell density optimization with cell mito stress test gt temp Your template files are stored in the Templates directory ap Templates gt XFe Assays n Designs XFe 7 15 2013 1 A ti j vath Dex ve aT XFe 7 15 2013 Gic obsis Anci Stress Test Note If you are not connected to an instrument upload your template file to a flash drive and transfer it to your instrument Open a Template in the Template Editor There are 2 ways to open a template in the Template Editor on the Desktop e Double click on the template file as
71. say design file Open Displays lists of recent analyses and design files and allows you to open an existing assay design file or analysis file Catalog Saves frequently used assay conditions to simplify assay design These conditions may include compounds used in your Injection Strategies as well as Pretreatments Media and Cell Types 12 Wave User s Guide Options Allows you to view customize and add default values for any instruments installed on your desktop or touch screen XFe24 XFe96 XFe24 Hypoxia Mode or XFe96 Hypoxia Mode The table below describes these advanced options Option Description Allows you to view and customize Login Settings Buffer Capacity Atmospheric Pressure Favorite Places maximum number of Recent Places Analysis Files and Design Files that appear when you click or touch Open Instrument Allows you to view and customize protocol defaults Advanced Allows you to specify the email addresses of people you would like to have notified when your assay has completed For more information on any of these options see Customizable Default Settings Help Provides the following information User s Guide The user s guide is searchable and has a table of contents from which you can click to a selected part of the guide You can also print it if you have a connection to a printer Support Support phone number Support email address and link to the Seahorse Bioscience
72. set the tolerance range If the temperature veers from the target temperature by too much the icon changes color e You can specify that the software send you a warning by email if the temperature exceeds the tolerance range The following example shows how the Temperature control icon looks if the temperature has exceeded the tolerance range Heater ON Note The Status Indicator on top of the XF Analyzer will also change from blue to amber if the temperature has exceeded the tolerance range For more information see the Status Indicator section To specify the target temperature and the tolerance range 1 Click on the Temperature icon It will display the Tray Temperature window as shown in the following example 44 Wave User s Guide ray Temperature Current 366 j C ig Heater On Tolerance Range C The Tray Temperature window shows the current temperature the target temperature and the tolerance range 2 Click on the up and down arrows to raise or lower the target temperature or type in a new number in the temperature field Target ah ET w 3 Click on the up and down arrows to raise or lower the tolerance range or type in a new number in this field Tolerance Range A o4 cC wv 5 Pressthe Save button To turn on an alarm that will be triggered if the temperature exceeds the tolerance range 1 Checkthe Alarm On box in the Temperature Control window v Alarm On 2
73. the barcode manually See the next section for details e Cancel to end your assay Entering the cartridge barcode manually If you click the Manual button from the Cartridge Barcode Error message the following dialog box is displayed 48 Wave User s Guide Barcode Information Lot Number Serial Number Cartridge Type O2 02 A O2 B pH PH A PH B PH C Cancel To complete this form 1 Enter the cartridge lot number in the Lot Number field The lot number is on the side of the cartridge labeled L N as shown in the example below 2 Enter the serial number in the Serial Number field The serial number is on the side of the cartridge labeled S N 3 Select the cartridge type from the Cartridge Type menu 49 Wave User s Guide 4 Enter the O2 values that are on the side of your cartridge in the O2_A and O2_B fields as shown in this example O2 A 10000 O2 B 0 022 O2 C 45000 pH A 35000 pH B 0 0025 pH C 10 5 Enterthe pH values that are on the side of your cartridge in the PH A PH B and PH C fields as shown in the previous example 6 Pressthe Accept button If you do not know all of these values 1 Openthe tray by pushing the Open Tray button Check the sides of the cartridge for the serial number O2 values and pH values Write down the serial number O2 values and pH values Press the Close Tray button Complete the Barcode Information form ES LEE E o Press the Accept bu
74. tions Allows you to view customize and add default values for any instruments installed on your desktop or touch screen XF 24 XF 96 XF 24 Hypoxia Mode or XF 96 Hypoxia Mode The table below describes these advanced options 13 Help Provides the following information c scscceccccececeesseseecceceeseeeeseseceeeeseseuaaseseeeess 13 XF Home Instrument Modes 13 Wave User s Guide Starta New Assay DGSIGM o oan n a 18 Define Groups and Conditions 18 Assay Conditions Injectiohs sssrinin A 19 Assay Conditions Ppretreatineftffffee 20 Assay Conditions XF Assay Medium 21 Assay Conditions Cell TVDB 22 Automatically Generate Groups 23 Manually Generate pp p p eee ee oer ae 24 Map Groups to the Plate Map 25 tj 9 n 0 25 Disteibule a esit pte 25 Backerouna ce E DO IE TUN PN 25 DEMING a PROTOCON nem 25 Default Commands and User Specified Injection Strategies 25 elder 26 Ee args Hog RITE TTE nm 26 Measurement CUBE c ine nn Re atte EE Pe nce esc op s E Ie er 26 WNYC CONS ces 28 OUD NAY srar 666 28 Save and Save ds Telfplaleu eon oe E ox dU Uis 29 Open a Template in the Template Editor
75. tton Plate barcode read failure If there is a plate barcode read failure you will receive the following message Plate barcode read failure Unable to read plate barcode Click Manual to enter the barcode manually or Cancel to cancel the Assay It provides 2 options e Manual to enter the barcode manually See the next section for details e Cancel to end your assay 50 Wave User s Guide Entering the plate barcode manually Plate Barcode Info Barcode Open Tray lose Tray Accep Cancel To complete this form 1 Enter the plate barcode in the Barcode field The plate barcode is on the side of the plate If you do not know the plate barcode 1 Openthe tray by pushing the Open Tray button Check the sides of the plate for the barcode Write down the number Press the Close Tray button Complete the Barcode Information form gx our doe T9 Press the Accept button Status Indicator When you are running an assay the Status Indicator light on top of the XF Analyzer will change color if you need to complete a task or if an error has occurred This LED changes from blue to amber to let you know if Wave has displayed any of the following questions or error messages e Asks you to load a cartridge or plate e Asks you to remove a used plate and cartridge e Asks you to accept or refuse a calibration after letting you know that one or more wells did not calibrate properly e Lets you know of an
76. ups to the Plate Map Defining a protocol in Define a Protocol Overview of the Process Design Run and Analyze There are three major steps to performing an experiment on the XF Analyzer design run and analyze This overview briefly explains each of these steps Step 1 Design your assay You can design your assay on your desktop or on the touch screen of your XF Analyzer Start T the Wave software by double clicking the shortcut on your desktop or by double tapping or d double clicking the shortcut on the touch screen of the XF Analyzer For comprehensive information on designing an assay refer to XF Home page Step 2 Run your assay You can design and analyze from your desktop computer however you have to transfer your assay to the XF Analyzer before running it To run a design created on the Wave software of the XFe Analyzer 1 Touch or click on the Review and Run tab of your touch screen 2 Touch or click the START RUN button To run a design created on your desktop if the XFe Analyzer is networked 1 Copy or move your design file asyd to your XF Analyzer 2 Log onto the Wave software on your XF Analyzer s touch screen by double tapping or f double clicking the shortcut Bes 3 Double tap or double click on the design file on the touch screen of your XF Analyzer If the Wave software is already open on the XF Analyzer click the Open command from your XF Home screen and Browse to the l
77. xcel 2007 2010 xlsx See Definitions for Templates Assay Design Files and Analysis Files for more information on these files r i 5 XV x gt m My Documents XFe Assays Search XFe Assays Organize Mew folder P m Name Date modified w Favorites BE Desktop 3 Cell density optimization with cell mito s 8 23 2013 3 08 PM ASYR File jg Downloads j GlycolysisAndStressTests 8 23 2013 310 PM ASYR File S Recent Places la mito glyco 8 23 2013 3 08 PM ASYR File BE Desktop Libraries Ej Documents al Music ai My Assays Pictures Bf Videos E I File name Cell density optimization with cell mito stress test 4 Select Excel 2007 2010 xlsx and browse to where you would like to save your Excel file 5 Click the Save button in the lower right hand corner File name LES TP 4 ET PT na Tes TERT E TL E C a CHR Hide Folders The following screenshot displays an example Excel spreadsheet that was generated by saving an analysis file in the Excel 2007 2010 xlxs format 10 Wave User s Guide B Cet density optimization with cell mito stress test ee 13 Piated On 2013 01 22114 00 00 34 Last Run 1 73 2013 4 44 76 PM 25 76 Cartridge Barcode 27 Cartridge Type 78 Cartridge Serial 23 Cartridge Lot 3 31 Mate barcode t Port C 25 pil 37 Fiate Type 33 Mate Senal 34 Plate Lot 35 Mate Orientation Notice that there are multiple ta
78. y errors that occur during the run including cartridge barcode errors plate barcode errors or protocol errors 51 Wave User s Guide Chapter 3 Managing Your Assays Wave provides several ways to manage your assays by giving you an easy way to e access your assay directories analyses and designs e access defined groups and conditions e customize default settings Access Directories Analyses and Designs Select the Open command under XF Home the My Places and My Assays panes that are displayed let you find your assays and designs XF Home New My Places My Assays Recent Assays Analyses Catalog amp Help mato glyco Options amp Help P My Assays Cell density optimization with cell mito stress test 4i Temp GlycolysisAndStressTests 4i Templates A XFe Assays ete Designs x SIR OG Cell Gensity m with SK mito Stress test New Xfe24 Assay XFe Cell Mito Stress Bn Browse My Places and My Assays My Places keeps track of the directories where you have stored or opened assays designs and templates If you click on Recent Assays My Assays or any other directory that you have visited while running Wave the assays and designs stored there will show up under the My Assays pane on the right hand side of the window 52 Wave User s Guide Recent Assays and My Assays are the default directories and are in color as shown in the following table Those directo
79. your XF Analyzer started the Wave software by 7 double clicking or double tapping the shortcut on your touch screen logged in and clicked on the Review and Run tab Wave will display the Review and Run window as shown in the example below Ff CUZ XFe Cell Mito Stress E3 A Cell density optimiza EJ d Sae 7 Group Definitions Instrument Protocol Review and Run otocc GENERAL Information Project Information Advanced Settings Project Name Well Volume u imal Notfcation Advanced On the right hand side of this window you will see a column of icons The following illustration provides a brief explanation of the purpose of these icons For more details read the next 4 sections 43 START RUN 0 0 C Heater ON The Tray icon allows you to insert or eject the tray Temperature Control 37 1 C Wave User s Guide Indicates whether your instrument is connected or not Indicates whether the heater is on or off and displays the tray temperature It also gives you control over the thermostat and heater Refer to the Temperature Control section for more information The Probes icon allows you to raise or lower the probes or load or unload the cartridge The Temperature licon serves as a thermometer and indicates whether the heater is on or off It also lets you have some control over the tray temperature e You can turn the heater on or off e You can set the target temperature e You can
80. yt to open the Template Editor siy e Alternatively right click on the template Ed icon on the XF Home gt New screen 32 Wave User s Guide Chapter 2 Reviewing and Running Your Assay Before you can run your assay on an XF Analyzer you must first move your design file or template file to the instrument if you did not design your assay on the instrument itself If your XF Analyzer is not networked copy your file to a USB flash drive then bring the flash drive to your XF Analyzer for transfer Note If you have a networked XF Analyzer you must close your design on the desktop before running it Log onto the Wave Software Log onto the Wave software on your XF Analyzer s touch screen by double tapping or double clicking the AF shortcut Ld then enter your username and password The first screen you see is the XF Home window From here you can open a design file or a template file Open a Design File To open an assay design file asyd and prepare to review it 1 Click on the Open command under XF Home 2 Click the Browse button circled in the example below to locate your design file fi XF Home My Places My Assays Recent Assays Analyses db Help mite abyca aii hiy Amas NW ralsrmhoenadin Cell derum aptimmation with cel mito stress Test i C j FRLIDOI i 4 ee Temp d Ciera a GhycohsisAnd Stress Tests ai Templates LE ERAT OUS FINIS m onam EPO ap SPE Sasaya C

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