Muse™ Bcl-2 Activation Dual Detection Kit User's Guide
Contents
1. ww POPULATION PROFILE Neg SETTING THE GATE To set the quadrant marker properly prepare a cell sample containing only the total antibody e g 5 uL of anti Bcl 2 PECy5 antibody 95 uL 1X Assay Buffer in a cell suspension Adjust the slider bars on the X and Y axis to place all populations Non expressing Inactivated and Activated on scale If the cell sample is not treated a great majority of the cell population will fall either in the Activated upper right or Non expressing quadrants lower left This is because Bcl 2 is inactivated by phosphorylation as indicated by Halder et al 1995 Adjust the quadrant markers to place the cell population s immediately to the right of the marker see diagram below Adjust the quadrant markers You can move the marker intersection in any direction as well as adjust the angle of each line To move the markers as they are touch the open circle at the intersection and drag the markers to make large changes or touch the arrow buttons below the plot to make small changes A To adjust the angle of either line touch the solid circle and drag in an arc or touch the arrow buttons below the plot B and C C Adjusting the Y axis Step 2 of 2 POPULATION PROFILE POPULATION PROFILE 4 4 5 jlnactivated Activated 2 jlnactivated Activated zy FA o x 9 7 e 7 oO ui Oo ha E a G 24 Q x x i2. see the Muse Cell Analyzer User s Guide 4 Fine tune the settings for the Bcl 2 EXPRESSION and CELL SIZE INDEX plot if necessary e Adjust the CELL SIZE INDEX slider accordingly to capture the cell population of interest see on screen instruction for example e Drag the threshold up or down to exclude cell debris Drag to make large changes Touch the arrow buttons located below the plot to make small changes The arrow buttons appear after you touch the threshold function NOTE If the acquisition times out after two minutes you can select Abort to restart the adjust settings step or Next to accept the settings and continue to the next step Adjust Settings Step 1 of 2 Adjust Settings Step 1 of 2 Pos P Capture cell population of 3 i interest by adjusting the i CELL SIZE INDEX slider bar on the X axis he cl 2 EXPRESSION i Bcl 2 EXPRESSION Touch threshold to Fine tune threshold LT activate and adjust 3 adjustments by 9 left right to exclude j using the arrows cell debris below 4 gt 4 m gt 1 Use slider to place cells in optimized region Bcl 2 EXPRESSION Bci 2 EXPRESSION 2 Drag threshold to eo exclude debris CELL SIZE INDEX Discard Changes 5 Select Next when you have completed the adjustments 6 Fine tune the settings for the Bcl 2 EXPRESSION vs PHOSPHORYLATION plot if necessary
3. Inactivated UL 22 01 Inactivated UL 92 00 22 01 920 Select plot to adjust marker gate POPULATION PROFILE 400 EXPRESSION Activated Activated UR Non expressing LL 5 00 9 97 50 Neg Bcl 2 EXPRESSION Pos inact PHOSPHORYLATION Act Figures A and B Jurkat cells were exposed to 1 uM staurosporine for 5 hours to induce cell death and illicit a Bcl 2 signaling cascade response the cells were then fixed permeabilized and stained with both anti phospho Bcl 2 Ser70 and anti Bcl 2 antibodies in multiplex Samples were acquired using the Muse Cell Analyzer and statistical results are shown above Figure A shows the results summary while Figure B shows results displayed by both dot plot and bar graph data The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population Cells which express Bcl 2 can be seen by the data on the top two quadrants of the dot plot inactivated and activated representing about 95 of the total cell population But of this cell population 92 is dephosphorylated upon treatment as indicated by a left shift confirming inactivation of the Bcl 2 signaling pathway upon treatment By presentation of both datasets one can now determine the total phospho ratio within their testing samples 13 Technical Tips 1 For cellular staining and analysis to be most effective make sure th
4. of the foregoing warranty EMD Millipore Corporation s sole obligation shall be to repair or replace at its option the applicable product or part thereof provided the customer notifies EMD Millipore Corporation promptly of any such breach If after exercising reasonable efforts EMD Millipore Corporation is unable to repair or replace the product or part then EDM Millipore shall refund to the Company all monies paid for such applicable Product EMD MILLIPORE CORPORATION SHALL NOT BE LIABLE FOR CONSEQUENTIAL INCIDENTAL SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS Unless otherwise stated in our catalog or other company documentation accompanying the product s our products are intended for research use only and are not to be used for any other purpose which includes but is not limited to unauthorized commercial uses in vitro diagnostic uses ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals c 2009 2012 Merck KGaA Darmstadt All rights reserved No part of these works may be reproduced in any form without permission in writing Cat No MCH200105MAN October 2012 Revision A
5. tab 12 Optional If changes are needed to the gates assigned touch the dot plot to enlarge it then adjust the cell size gate according as described in steps 4 and 6 respectively You cannot adjust the 002 Sample 002 cell size threshold after the sample has been acquired If you adjust the gate on subsequent samples and wish to apply the changes to other samples that you already acquired select the Apply Changes button in the title bar Select the samples you want to apply the changes to or choose Select All then select Apply The sample you originally made changes to must be selected Marker POPULATION PROFILE Select to apply 4 Inactivated 92 00 96 Ld Bcl 2 EXPRESSION 29 Non expressing EE changes to 3 00 other samples Inact 1 2 PHOSPHORYLATIO Act The quadrant marker is set oa Woemraracl statistics of the cellular pop 1 desired location 4 Back Return to Results 13 If no adjustments are needed select Next Run and repeat steps 9 through 12 for the remaining samples NOTE During the run a message may appear prompting you to load a tube of DI water for a Quick Clean Load the water then select Clean to perform the Quick Clean Select Next to continue with the run The frequency of Quick Cleans was set by your system administrator Your administrator may also have chosen to allow you to skip the Quick Clean when the prompt ap
6. Muse Bcl 2 Activation Dual Detection Kit User s Guide Catalog No MCH200105 50 tests FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA amp Canada Phone 1 800 437 7500 Fax 1 951 676 9209 Australia 61 3 9839 2000 www millipore com Introduction EMD Millipore s Muse Dual Detection kits are a series of products which include a pair of antibodies that bind to the same protein one to detect total protein expression and another to detect the phosphorylated form of the same target So by using two parameter analysis we can achieve target specific detection of phosphorylation and by doing so eliminate false positives while enhancing the signal to noise ratio Data generated using the Muse Cell Analyzer with the Muse software provides e Percentage of inactivated cells e Percentage of activated cells via phosphorylation e Percentage of non expressing cells The signaling pathways that regulate apoptosis are important in understanding tissue homeostasis the development of the immune system and therapies for cancer Bcl 2 is a member of the Bcl 2 family that regulates apoptosis by regulating the proteins that control mitochondrial membrane permeability 1 2 The Bcl 2 family consists of two protein sub groups the anti apoptotic proteins and the pro apoptotic proteins 3 Bcl 2 is an anti apoptotic family member that binds to pro apoptotic family members such as Bax to sequester them and inhibit ins
7. as been optimized to function for multiple cells types every cell line behaves differently The wide peaks or false peak may indicate that e The sample is poorly fixed and stained as a result of cell aggregates Ensure your sample is a single cell suspension before fixing and staining e Cell concentration is too high Decrease the number of cells by diluting the sample to 300 700 cells uL The Muse Cell Analyzer gives the most accurate data when the flow rate is between 300 and 700 cells uL Low level of staining e Although the assay procedure has been optimized to function utilizing multiple cell types every cell line behaves differently A low signal may indicate that the cells need to be stained at a higher volume e Verify that the System Check procedure was performed and the results passed Variability in day to day experiments e lf the results are inconsistent check that the samples were well mixed prior to acquisition Cells may quickly settle in your samples and your results will be inaccurate unless the cells are mixed just prior to acquisition e Monitor experimental cell cultures to ensure that cell viability and cell numbers being analyzed are consistent Any drop in cell numbers or viability can influence experimental results e f there appears to be day to day variation of the staining pattern ensure the Muse Cell Analyzer is working properly Check the Muse System Check log to ensure day to day
8. at test cells have good viability prior to use 2 For certain cell cultures cell pellets may become hazy or transparent following the fixation step making it difficult to see If sampling a small collection of cells for flow analysis it is recommended that all steps be performed in a smaller collection tube e g centrifuge tube 3 Do not mix or interchange reagents from various kit lots 4 Mix each cell sample thoroughly on a mixer before acquiring samples for consistent and accurate results However avoid vigorous mixing which can cause cellular breakdown and splashing resulting in volume loss and erroneous results 5 The default number of events to acquire is 1000 events You may select a different number however your statistical error will increase as you decrease the number of acquisition events 6 If results deviate from expected values prepare a freshly stained sample and reacquire the data 7 Periodically run Quick Clean using a tube of DI water after every 20 sample acquisitions to prevent a buildup from cellular debris in the system If your samples contain significant amounts of cellular debris run the Quick Clean cycle more often to prevent clogs or blockage 8 If you are acquiring data from a sample but the progress bar is not moving there is probably either insufficient volume to continue to acquire the sample or a blockage of the flow system First check to ensure that there is at least 100 uL of sample in the t
9. ents included in the kit may be harmful Please refer to the MSDS sheet for specific information on hazardous materials MSDS forms can be found on the web page or by contacting Millipore technical services e During storage and shipment the directly conjugated antibodies may condense within the vial For maximum recovery of the product centrifuge original vial prior to removing cap e The conjugated antibody is light sensitive and must be stored in the dark at 2 8C e Do not use reagents beyond the expiration date of the kit Storage All reagents must be stored at 2 8 C All kit components are stable up to four 4 months from date of receipt if stored and handled correctly Please avoid repeated changes in temperature as this will affect the integrity of the product Before You Begin It is highly recommended that you run the cell samples shortly after completing the sample preparation While some cell types have been shown to yield stable results for up to 24 hours after cell fixation permeabilization antibody staining if properly stored but the stability of individual cell types may vary Time considerations When dealing with phospho specific activation detection fixation of cell samples after cell treatment s is critical to capture the phosphorylation activation event Some activation state cell signaling responses are transient and may be lost if cell cultures are not fixed immediately following treatment Cell fixati
10. ertion into the mitochondrial membrane 4 5 When cells receive an apoptotic signal Bcl 2 releases Bax allowing Bax to form a complex on the mitochondrial membrane that releases cytochrome C into the cytoplasm leading to caspase activation and cell death 4 Bcl 2 has been shown to be regulated at the level of phosphorylation by JNK at a multi site phosphorylation loop which includes serine 70 6 Phosphorylation of Bcl 2 at Serine 70 has been shown to be important in both apoptosis and in autophagy 6 7 8 9 All Muse Dual Detection kits are optimized on the Muse Cell Analyzer Both antibodies provided in the kit are carefully titrated and optimized together to ensure maximal performance when run in multiplex alleviating the need for any additional optimization This kit contains optimized fixation permeabilization and assay buffers to provide researchers with a complete solution for Bcl 2 signaling analysis Test Principle The Muse Bcl 2 Activation Dual Detection Kit includes two directly conjugated antibodies a phospho specific anti phospho Bcl 2 Ser70 Alexa Fluor 555 and an anti Bcl 2 PECy5 conjugated antibody to measure total levels of Bcl 2 expression This two color kit is designed to detect the extent of Bcl 2 pathway activation by measuring Bcl 2 phosphorylation relative to the total Bcl 2 expression in any given cell population By doing such the levels of both the total and phosphorylated protein can be measured simul
11. f the antibody working cocktail solution as previously described to each microcentrifuge tube containing the cell suspension 14 Incubate cell testing samples for 30 minutes in the dark at room temperature 15 Following incubation step add 100 uL of 1x Assay Buffer to each microcentrifuge testing sample and centrifuge at 300 x g for 5 minutes on a tabletop centrifuge Discard supernatant 16 Resuspend cells in each microcentrifuge tube with 200 uL of 1x Assay Buffer 17 Acquire samples on the Muse Cell Analyzer using the onscreen instructions Setup and Acquisition on the Muse Cell Analyzer Run a System Check prior to performing the assay For information on Muse System Check refer to the Muse Cell Analyzer User s Guide 1 Select Bcl 2 Activation from the main menu All Assay Search Filter Annexin V amp Dead Cell Bcl 2 Activation D Caspase 3 7 Li CelOme Cell Cycle 2 Select Run Assay Home Assay Settings Run Results Bcl 2 Activation Run Assay View Results 3 Adjust the instrument settings e Load the sample for adjusting the settings and select Run Note Perform the adjust settings step using a negative control e g total antibody then verify the settings using a positive control Mix and Load Raise e Or to retrieve previously saved instrument CC ae SHEHUT settings select Retrieve Settings For more information on retrieving settings
12. instrument variation is low 15 References 1 Cory S et al 2002 The Bcl2 family regulators of the cellular life or death switch Nat Rev Cancer 2 9 647 56 2 Adams J M et al 1998 The Bcl 2 protein family arbiters of cell survival Science 281 5381 1322 6 3 Huang D C et al 2000 BH3 Only proteins essential initiators of apoptotic cell death Cell 103 6 839 42 4 Gross A et al 1998 Enforced dimerization of BAX results in its translocation mitochondrial dysfunction and apoptosis EMBO J 17 14 3878 85 5 Yang E et al 1995 Bad a heterodimeric partner for Bcl XL and Bcl 2 displaces Bax and promotes cell death Cell 80 2 285 91 6 Wei Y et al 2008 Dual role of JNK1 mediated phosphorylation of Bcl 2 in autophagy and apoptosis regulation Autophagy 4 7 949 51 7 Ito T et al 1997 Bcl 2 phosphorylation required for anti apoptosis function J Biol Chem 272 18 11671 3 8 Haldar S et al 1995 Inactivation of Bcl 2 by phosphorylation Proc Natl Acad Sci USA 92 10 4507 11 9 Haldar S et al 1996 Taxol induces bcl 2 phosphorylation and death of prostate cancer cells Cancer Res 56 6 1253 5 Related Products Muse H2A X Activation Dual Detection Kit Catalog No MCH200101 Muse EGFR RTK Activation Dual Detection Kit Catalog No MCH200102 Muse PI3K Activation Dual Detection Kit Catalog No MCH200103 Muse MAPK Activation Dua
13. king longer than expected or progress bar stops during acquisition Ensure that the System Check procedure was run and passed If the progress bar stops during acquisition the fluid system may be clogged Run a Quick Clean procedure to clean the capillary It can be performed during or after an assay Instrument clogging If the instrument is clogged run a Quick Clean procedure to clean the capillary It can be performed at anytime during an assay between samples No detectable phosphorylation activation in testing samples Since phospho specific activation can be very quick transient responses in order to capture this phosphorylation event samples must be immediately fixed to freeze the given activation state in time Low Cell Concentration warning during Ensure that cells are counted properly prior to beginning the experiment The assay instructions are optimized to give you a range of cells between 300 700 cells uL in the final sample volume so accurate population count aesgismen results are obtained A substantial decrease in cell numbers can lead to difficulty in adjusting settings High Cell If the concentration of the stained cell sample is high 21200 cells uL dilute Concentration the sample further with Muse Cell Cycle Reagent to adjust the cell warning during concentration between 300 and 700 cells uL acquisition High CVs wide peaks or false peak Although the assay procedure h
14. l Detection Kit Catalog No MCH200104 Muse System Check Kit Catalog No MCH100101 Muse Count amp Viability Kit 100T Catalog No MCH100102 Muse Count amp Viability Kit 600T Catalog No MCH600103 Muse Count amp Viability 200X Catalog No MCH100104 9 Muse Annexin V amp Dead Cell Kit Catalog No MCH100105 10 Muse Cell Cycle Kit Catalog No MCH100106 11 Muse Caspase 3 7 Kit Catalog No MCH100108 12 Muse MultiCaspase Kit Catalog No MCH100109 13 Muse Mitopotential Kit Catalog No MCH100110 14 Muse Cell Dispersal Reagent Catalog No MCH100107 Oo OPE des eee Warranty EMD Millipore Corporation EMD Millipore warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products EMD MILLIPORE MAKES NO OTHER WARRANTY EXPRESSED OR IMPLIED THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE The warranty provided herein and the data specifications and descriptions of EMD Millipore products appearing in EMD Millipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of EMD Millipore Representations oral or written which are inconsistent with this warranty or such publications are not authorized and if given should not be relied upon In the event of a breach
15. lowed by a washing step i Centrifuge cells at Add 200 000 cells to each tube 300 x g for 5 min treated or untreated and wash with 1X Assay Buffer Kit Components e 20X Anti phospho Bcl 2 Ser70 Alexa Fluor8555 Part No CS208208 One vial containing 250 uL e 20X Anti Bcl 2 PECy5 Part No CS208209 One vial containing 300 uL e 5X Assay Buffer Part No CS202124 One bottle containing 55 mL e Fixation Buffer Part No CS202122 One bottle containing 13 mL e Permeabilization Buffer Part No CS202125 One bottle containing 13 mL Materials Not Supplied Test tubes for sample preparation and storage Tissue culture reagents i e HBSS PBS w o Ca or Mg cell dislodging buffers etc Pipettes with corresponding tips capable of accurately measuring 10 1000 uL Tabletop centrifuge capable of achieving 300 x g Mechanical vortex Deionized Water for buffer dilution Cells of interest in suspension e g Jurkat po 2 GO SUME de Om d oem Microcentrifuge tubes with screw caps 1 5 mL VWR Catalog No 16466 030 or equivalent 9 Muse Cell Analyzer 10 Muse System Check Kit Catalog No MCH100101 11 Guava ICF Instrument Cleaning Fluid Catalog No 4200 0140 optional Precautions e The instructions provided have been designed to optimize the kit s performance Deviation from the kit s instructions may result in suboptimal performance and may produce inaccurate data e Some assay compon
16. on permeabilization and staining will take approximately 50 minutes Acquiring data on your Muse Cell Analyzer takes less than 3 minutes per sample depending on the cell concentration and desired number of events to acquire Always perform a System Check prior to performing the assay For details refer to the Muse Cell Analyzer User s Guide Preparation of Reagents 1 Assay Buffer Assay Buffer is supplied at 5X concentration and should be diluted to 1X with deionized water prior to use Prepared 1X Assay Buffer is stable up to one year Store at 2 8 C 2 Antibody Working Cocktail Solution The kit contains two 2 antibodies which can be used in multiplex Prior to antibody staining of cells prepare an antibody working cocktail solution by addition of the following Add 5 uL of anti phospho Bcl 2 Ser70 Alexa Fluor 555 and 5 uL of anti Bcl 2 PECy5 conjugated antibodies into a centrifuge tube for a final volume of 10 uL total This amount is good for one 1 test Based on the number of tests tubes being performed it is up to the end user to adjust antibody volume amounts at similar ratios e g for 10 tests the working cocktail solution will contain 50 uL of anti phospho Bcl 2 Ser70 and 50 uL of anti Bcl 2 for a total of 100 uL Aliquot 10 uL of the working cocktail solution per test tube sample This solution should be prepared as needed but if temporary storage is needed please keep in the dark at 2 8C Assay Inst
17. on the instrument loading arm Select Run to acquire the sample Run Sample y Running During acquisition live statistical values are generated and plotted onto a bar graph Measurements POPULATION PROFILE as EXPRESSION 36 include inactivated activated and A EE non expressing cells Mix and Load Raise Sample Sample Loader Select Run 1 Hi Neg Bcl2 EXPRESSION Pos Abort Stop amp discard run 11 When acquisition is complete the results are displayed If desired select Plots to display a dot plot and a bar graph for the sample Home Assay Settings Run Results Home Assay Settings Run Options Clean z Results Options Clean 002 Sample 002 v a 002 Sample_002 lt Results Statistics EEWO Statistics EEU Ini of Cells Total Mean ofCells Intensity Inactivated UL Select to hide plots Select plot to adjust marker gate Activated UR POPULATION PROFILE Non expressing LL Neg Bcl 2 EXPRESSION Pos Inact PHOSPHORYLATION Act Finish Next Run Select to display Finish Next Run Save amp close data set Run next sample plots Save amp close data set Run next sample You can view or change the sample ID as well as add annotations for the current sample by selecting the Sample Info Tab To print the results for the current sample select the printer
18. pears You can choose to perform additional Quick Cleans at any time during a run by selecting Clean in the title bar then Quick Clean from the menu 14 When you have acquired the last sample select Finish 15 Optional Select Options in the title bar to rename the data set export the data set save the current instrument settings or view the event log Refer to the Muse Cell Analyzer User s Guide for more information 12 Quick Clean Load sample tube containing DI Water ample Loa Dr Bi elect laar Results The software performs calculations and displays the data in two plots e A dot plot displaying cells which are inactivated non expressing and activated on a bivariate plot indicating Bcl 2 expression and phosphorylation for a given testing sample e A bar graph illustration of the same data calculating the expression of each cell population as a percentage 9 Results from each run are stored in a data file as well as its corresponding spreadsheet CSV file The data file and spreadsheet file contain the following statistics e Sample Number e Sample ID e Percent totals for the Inactivated Activated Non expressing cell types e Mean Fluorescence Intensity for the Inactivated Activated Non expressing cell types Statistics EWIE Statistics Sample Info Mean Intensity Mean Intensity Bcl 2 Total of Cells Bcl 2 Total of Cells
19. ructions Note This assay protocol has been optimized using human Jurkat cells However this kit is suitable for measuring the extent of Bcl 2 target specific detection of activation via phosphorylation on a variety of human cell types in which Bcl 2 is expressed Alternate species reactivity must be confirmed by the end user I Cell Culture and Stimulation Used for example purposes 1 Prepare cells of interest into two tissue culture flasks treated or untreated overnight in a 37 C incubator with 596 CO2 Cells should be at about 9096 confluent the next day 2 For the flask labeled Treated treat cells accordingly e g chemically treated using staurosporine or compound of choice The intent is to induce Bcl 2 activation for the given cell type The other flask labeled untreated will serve as a control 3 Incubate the flasks in a 37 incubator with 5 CO2 Exposure time and treatment concentrations are determined at the discretion of the end user 4 Deactivate cells by exchanging out the growth media with fresh growth media or 1X PBS All cell treatments and experimental samples are determined by the end user This section is provided only as an example for inducing a Bcl 2 response for measurement of phospho Bcl 2 activation ll Fix and Permeabilize Cells 5 After cellular deactivation spin down the treated and untreated testing samples at 300 x g for 5 minutes and discard the media 6 Resuspend cell
20. s by adding 500 pL of 1X Assay Buffer per one million cells for larger cell samples i e 5x10 cells add 2 5 mL 1X Assay Buffer to cell sample Essentially add 50 uL of 1X Assay Buffer for every 100 000 cells evaluated 7 Add equal parts Fixation Buffer to cell suspension 1 1 So for every 500 uL of 1X Assay Buffer per one million cells add an additional 500 uL Fixation Buffer for a total of 1 mL cell fixation solution and mix sample by gently pipetting up and down Similarly add 50 uL of Fixation Buffer for every extra 100 000 cells evaluated to keep the 1 1 ratio consistent Incubate for 5 minutes on ice 8 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 9 Permeabilize cells by adding 1 mL ice cold Permeabilization Buffer per one million cells and incubate on ice for 5 minutes For smaller cell samples i e 500 000 cells add 500 uL ice cold Permeabilization Buffer 10 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 11 Resuspend cells in 450 uL 1X Assay Buffer per one million cells and aliquot 90 uL per microcentrifuge tube Compatible for the Muse Cell Analyzer Please see Materials Not Supplied section on page 3 Ill Cell Staining and Analysis 12 For single color staining e g adjustment settings add 5 uL of anti Bcl 2 PECy5 antibody to the microcentrifuge tube containing the cell suspension 13 For multiplexing add 10 uL o
21. taneously in the same cell resulting in a normalized and accurate measurement of Bcl 2 activation after treatment Moreover simultaneous measurement of both total and phospho Bcl 2 confirms target specificity of the phosphorylation event Together a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho total ratio within a mixed cell population The antibody pair provided in the kit has been carefully titrated to ensure the ability to measure total and phospho Bcl 2 simultaneously on the same protein for accurate determination of protein level and activation Sufficient reagents are provided to perform 50 tests Detailed assay instructions are included to assist in analysis and to ensure the correct cell concentration is obtained during acquisition of sample data For Research Use Only Not for use in diagnostic procedures Summary of Protocol Prepare cell cultures for experimentation dd 10 uL of the antibody treated or untreated cocktail to 90 uL of 1X Resuspend in 200 uL 1X ssay Buffer per tube test Assay Buffer and Centrifuge cells at 300 x g for 5 min and low to incubate for 30 min acquire samples on wash once with 1X PBS at room temp dark Muse Cell Analyzer Fix cells in Fixation Buffer for 5 min on ice followed by a washing step rT P bilize cells with P bilizati ermeabilize cells with Permeabilization gt amp d Buffer for 5 min on ice fol
22. ube If not add additional buffer to bring the volume up to 100 uL or proceed to the next sample If the sample volume is greater than 100 uL then the lack of events is probably due to a clog A clog or blockage can be caused by cell aggregates cell debris bleach crystals or other particulates Perform a backflush to flush out the clog into a tube containing 20 bleach Then run Quick Clean to remove bleach residue If this procedure does not alleviate the problem refer to the Muse Cell Analyzer User s Guide for additional troubleshooting tips or contact Millipore Technical Support for help 9 The Muse Bcl 2 Activation Dual Detection Kit works best with samples in single cell suspension Cell aggregates may clog or be excluded from the flow cell affecting the accuracy of the results If you wish to use the Muse Bcl 2 Activation Dual Detection Kit with a clumpy cell line it is recommended to order Muse Cell Dispersal Reagent Catalog No MCH100107 to for detailed information on the Muse Cell Dispersal Reagent and assay method For more troubleshooting tips refer to the Muse Cell Analyzer User s Guide For more information contact the Millipore office nearest you In the USA call 1 800 MILLIPORE 1 worldwide contact information You can also view the tech service page on our website at www millipore com techservice 14 Troubleshooting Potential Problem Experimental Suggestions Acquisition ta
23. w 7 P q o 5 e a a 0 Non expressing 3 o Non expressing 0 Non expressing 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Inact PHOSPHORYLATION Act A inact PHOSPHORYLATION Act mA Inact PHOSPHORYLATION Act POPULATION PROFILE POPULATION PROFILE 1 To set conirols place 1 To set i BI 1 To set i place eq place cq q 2 Move gd 2 Move gi c populati ed populatii eq Non exq Non exq 7 Select Next when the marker adjustments are complete 8 Verify the settings If the settings are correct select Next Otherwise select Back and repeat steps 4 through 7 as necessary Verify Settings POPULATION PROFILE Inactivated Activated Ww Neg Bcl 2 EXPRESSION Pos N Neg Bel 2 EXPRESSION Pos N Non expregsing o 1 2 3 0 1 2 3 4 CELL SIZE INDEX inset PHOSPHORYLATION Act If settings are correct select Next otherwise select Back Back Set Population Profile 9 Enter the sample ID by touching the field then using the keypad to input the ID Touch Done when you re finished entering the ID If necessary change the Events to Acquire by touching the field then selecting the value from the pop up menu Select Next Home Assay Settings Run Results Sample Info Clean w Sample 001 Sample ID Sample 001 26 Events to Acquire 1000 Events v 10 10 Follow the onscreen instructions and mix the first sample Load the sample
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