Muse™ Autophagy LC3-antibody based Kit User's Guide
Contents
1. eee Running M During acquisition live statistical 3 AUTOPHAGY INDUCTION PROFLE values are generated and represented on a histogram graph 4 POPULATION PROFILE CELL SIZE INDEX LC3 INTENSITY Next Skip rest of events 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 Results 002 Starvation M Statistics Relais Mean Autophagy Intensity Total of Cells Autophagy LC3 detection Finish Next Run Save amp close Data Set Run next sample a Home Assay Settings Run Results Results 002 Starvation a Statistics REE aLe Mean Autophagy Autophagy Induction Intensity Ratio Total of Cells Autophagy LC3 detecti etection Select to hide Select plot to adjust marker gate plots POPULATION PROFILE AUTOPHAGY INDUCTION PROFILE CELL SIZE INDEX 1 2 3 LC3 INTENSITY Low 2 3 Hig n4 AUTOPHAGY INTENSITY Finish Next Run 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 tab 11 12 Optional If changes are needed to the gates assigned touch the histogram plot to enlarge it then adjust the histog2. Muse Autophagy LC3 antibody based Kit User s Guide Catalog No MCH200109 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA amp Canada Phone 1 800 645 5476 In Europe please contact Customer Service France 0825 045 645 Spain 901 516 645 Option 1 Germany 01805 045 645 Italy 848 845 645 United Kingdom 0870 900 46 45 For other locations across the world please visit www millipore com offices Introduction Autophagy is an intracellular catabolic pathway that causes cellular protein and organelle turnover and it is associated in diverse diseases including Alzheimer s disease aging cancers and Crohn s disease lt is a tightly regulated process that plays a normal part in cell growth development and cellular homeostasis Autophagy functions as a housekeeping mechanism by disposing of aging and or dysfunctional proteins and organelles through sequestering and priming proteins for lysosomal degradation Increasing evidence suggests that not just apoptosis but also autophagy can contribute to cell death and greatly influence general cell health Malfunctions of the autophagy process are proposed to influence cell health longevity and the capability of cells to function at full capacity EMD Millipore s Muse Autophagy LC3 antibody based kit provides a quantitative solution for the study of autophagy This kit contains two key detection reagents to help facilitate the monitoring of lipidated LC3 II in
3. 6 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 Autophagy LC3 detection from the main menu Home Assay Settings Run Results WEN Muse Seup gt Favorites Count amp Viability Annexin V amp Dead Cell Cell Cycle SmartFlare Detection ao gt La Autophagy LC3 detection d For Research Use Only Not for use in diagnostic procedures 2 Select Run Assay Home Assay Settings Run Results Autophagy LC3 detection Meee ie 5 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 no treatment no starvation then verify the settings using a positive control e g treated starvation e Or to retrieve previously saved instrument Mix and Load Raise n Sample sampe Loader settings select Retrieve Settings For more information on retrieving settings see the Muse Cell Analyzer User s Guide 4 Fine tune the settings for the AUTOPHAGY INTENSITY 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 c
4. ATION PROFILE AUTOPHAGY INDUCTION PROFILE gt lt ii e m N 77 E iT O LC3 INTENSITY AUTOPHAGY INTENSITY Figures A and B HeLa cells were either starved for 4 hours to induce autophagy or kept under fed conditions and then stained with the anti LC3 Alexa Fluor amp 555 conjugated antibody Samples were acquired using the Muse Cell Analyzer and statistical results are shown above Figure A shows the results summary for the test sample as well as the corresponding histogram plot comparing the control versus the target sample The statistics captured in this assay can also be illustrated by a data results summary table as shown in B Here the mean autophagy intensity for each sample is determined and the autophagy induction ratio is then calculated based on the ratio between the target sample fluorescence versus the control sample In this cell population there is a 6 4 fold change between the control sample e g no autophagy in blue when compared to the starved sample e g induced autophagy in red indicating the presence of autophagy 13 Technical Tips 1 Confirm that testing cells are greater than 80 viability prior to assay 2 For drug treatments and or starvation times all incubation times and sample concentrations must be optimized at the researcher s own discretion to suit the researcher s individual experimental needs 3 Do not mix or interchange reagents from various kit lots 4 Mix each cell sampl
5. a given cell system gt The use of selective permeabilization solution discriminates between cytosolic LC3 from autophagic LC3 by extracting the soluble cytosolic proteins while protecting LC3 which has been sequestered into the autophagosome gt Since autophagy is a constitutive cellular degradation process the use of an autophagy detection reagent Autophagy Reagent A will prevent the lysosomal degradation of LC3 allowing its quantification by flow cytometry Data generated using the Muse Cell Analyzer along with the corresponding Muse software module provides statistical values measuring e Mean Autophagy Value for both control and test samples e Autophagy Induction Ratio Test sample fluorescence relative to control e Percentage of cells with increased autophagy Test sample versus control By having the ability to measure and quantify autophagy one can screen autophagy inducers or inhibitors monitor cell culture health and protein turnover rate study the mechanisms of protein degradation and identity new autophagy targets and pathways leading to aging and neurodegenerative diseases The Muse Autophagy LC3 antibody based kit is optimized on the Muse Cell Analyzer The anti LC3 Alexa Fluor amp 555 conjugated antibody and autophagy enabling reagents have been carefully evaluated to ensure optimal performance alleviating the need for any additional validation of the kit reagents This kit contains reagents A and B
6. along with an assay buffer to provide researchers a complete solution for autophagy analysis Mutrient Depletion Cytosolic Proteins feo LOS GB Aging Organelles i Cytosolic Proteins 1 Induction and LC3 translocation for breakdown 2 Autophagosome formation 3 Docking amp fusion with the lysosyme Autophagosome VS050MC 4 Autophagosome breakdown Figure 1 Autophagy Four Stages of Autophagy Autophagy can be induced by nutrient depletion or inhibition of mTOR pathway During autophagy cytosolic proteins and aging organelles are sequestered by a double membrane vesicle to form autophagosomes One of the hallmarks of autophagy is translocation of LC3 from the cytoplasm to the autophagosome Autophagosome then fuses with the lysosome to cause the breakdown of autophagosome vesicle and its contents including LC3 This process can be visualized using a RFP LC3 fusion protein Product Overview Discrimination between cytosolic and autophagosome associated LC3 is achieved by monitoring the translocation of LC3 using flow cytometry This kit provides the reagent for the disruption of the cell plasma membrane using a proprietary selective permeabilization solution figure 2 The selective permeabilization solution will extract cytosolic LC3 by flushing away during washing steps LC3 translocated into the autophagosome is protected from the extraction and remains intact inside autophagosome thereby allowin
7. ated cells remove growth media from cells and wash cells once with 1X HBSS Replace media with 200 uL of EBSS 0 2 uL Autophagy Reagent A 1 1000 dilution at 37 incubator for 276 hours to induce autophagy under starvation conditions for untreated cells continue culture with growth media supplemented with FBS in the 96 well plate After 2 6 hours treatment aspirate wells to remove culture supernatants and wash the cells once with 1X HBSS Detach cells with 100 uL Accutase or any other mild enzyme for 5 minutes at 37 C Transfer cells to a Muse compatib le sample tube s and add 100 uL 1X HBSS to each well Spin the sample tubes at 300 x g for 5 minutes at 4 C and remove supernatant Add 5 uL of Anti LC3 Alexa Fluor 555 and 95uL of 1X Autophagy Reagent B to each sample tube Stain on ice for 30 minutes in the dark Spin the sample tubes at 300 x g for 5 minutes at 4 C and remove supernatant then wash cells once with 1X Assay Buffer Resuspend each sample tube in 200 uL 1X Assay Buffer and acquire by Muse immediately During cellular acquisition event counts per uL should not exceed 800 Check for counts per uL when adjusting settings Dilute your sample to target 500 events per uL Note LC3 expression can vary depending on the cell line being evaluated The kit is designed to measure endogenous levels of LC3 so be aware that some cell types may have very low endogenous levels of LC3 not measureable by immunodetection methods
8. blications are not authorized and if given should not be relied upon In the event of a breach 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 EMD 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 2013 Merck KGaA Darmstadt All rights reserved No part of these works may be reproduced in any form without permission in writing 17 Catalog No MCH200109 May 2013 Revision A MCH200109MAN
9. cad Sci U S A 104 48 19023 8 Degtyarev M et al 2008 Akt inhibition promotes autophagy and sensitizes PTEN null tumors to lysosomotropic agents J Cell Biol 183 1 101 16 Mizushima N et al 2004 Methods for monitoring autophagy Int J Biochem Cell Biol 36 12 2491 502 Fleming A et al 2010 Chemical modulators of autophagy as biological probes and potential therapeutics Nat Chem Biol 7 1 9 17 Tian Y et al 2011 A small molecule enhancer of autophagy decreases levels of Ap and APP CTF via Atg5 dependent autophagy pathway FASEB J 25 6 1934 42 Turcotte T et al 2008 A molecule targeting VHL deficient Renal Cell Carcinoma that induces autophagy Cancer Cell 14 1 90 102 16 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 MERCHANT ABILITY 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 pu
10. ditional troubleshooting tips or contact Millipore Technical Support for help 8 The Muse Autophagy LC3 antibody based 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 cell cultures continue to be clumpy it is recommended to order Muse M Cell Dispersal Reagent Catalog No MCH100107 to disaggregate the cells Contact customer service or visit our website at www millipore com muse 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 1 800 645 5476 Outside the USA visit our website at www millipore com offices for up to date worldwide contact information You can also view the tech service page on our website at www millipore com techservice 14 Troubleshooting Potential Problems Experimental Suggestions Acquisition taking longer than expected or progress bar stops during acquisition Instrument clogging No detectable levels of autophagy in testing samples Low cell concentration warning during acquisition High cell concentration warning during acquisition High CVs wide peaks or false peak Ensure that the System Check procedure was run and passed If the progress bar stops during acquisition the fl
11. e 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 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 7 f 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 tube 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 ad
12. ell debris Drag the bar 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 four minutes you can select Abort to restart the adjust settings step or Next to accept the settings and continue to the next step j Capture cell population of Adjust Settings Step 1 of 2 interest by adjusting the CELL SIZE INDEX slider bar on the Y axis CELL SIZE INDEX Touch threshold to activate and adjust up down to exclude cell debris 0 1 2 3 E Low AUTOPHAGY INTENSITY High m E b POPULATION PROFILE POPULATION PROFILE 1 Use in op 1 Use sliders to place cells in optimized region 2 Drag Threshold to exclude Debris e wW y a a HR w 5 AUT Discard Changes Fine tune threshold adjustments by using the arrows below 5 oelect Next when you have completed the adjustments 6 Fine tune the settings for the LC3 INTENSITY histogram plot if necessary e SEITING THE HISTOGRAM MARKER To set the histogram marker properly prepare a control cell sample e g no treatment and fed no autophagy and place the histogram marker immediately to the left edge of the peak as shown in A This will represent your negative cells e Adjust and extend the other end of the histogram marker to the right to cover the region to the right of this control sample histog
13. file 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 Note It is very important to first run a control sample prior to acquisition of the experimental target samples Data analysis and result values are based on the autophagy induction ratio where the mean autophagy intensity of the target sample is compared relative to the control sample negative i First Sample Control Home Assay Settings Results Sample Info Clean F Sample 001 Sample ID Sample 001 X Events to Acquire 1000 Events v Next Load oample When first running the first sample ii Subsequent Sample s Target Home Assay Settings Results ample Info Clean F Sample 002 Sample ID Sample 002 Events to Acquire 1000 Events v make sure that the control in the field sample type is below Subsequent samples will be experimental samples Make sure that sample type is identified as target 10 10 Follow the onscreen instructions and mix the first sample Load the sample on the instrument loading arm Select Run to acquire the sample Run Sample Mix and Load Raise Sample Sample Loader Select Run Y O Run Run Sample
14. g its fluorescence to be measured by flow cytometry or imaging oince autophagy is a constitutive cellular degradation process the use of an autophagy detection reagent Autophagy Reagent A will prevent the lysosomal degradation of LC3 allowing for quantification of its fluorescence Accumulation of Autophagosomes Figure 2 Selective Permeabilization helps discriminate cytosolic from autophagic LC3 Discrimination between cytosolic LC3 I from autophagosome associated LC3 II is achieved by disruption of cell PM by using an autophagy Selective Selective Permeabilizztion Permeabilization enabling solution Autophagy Reagent B i i This selective permeabilization will release cytosolic LC3 by flushing away during washing steps LC3 II trapped in 1 the autophagosome remains intact and fluorescence can A ff be measured NM d E NA m 7 mm d F e fm a n M Londen EMD Mlillipore s Muse Autophagy LC3 antibody based kit includes an anti LC3 mouse monoclonal antibody conjugated to Alexa Fluor amp 555 used to measure and track the levels of LC3 within the cell The autophagy detection reagents and antibody have been optimized together to ensure the ability to measure and discriminate between cytosolic and lipidated LC3 to accurately measure the autophagic process Sufficient enabling reagents are provided to perform 100 tests Detailed assay instructions are included to assist in analysis For Research Use Only Not f
15. iluting 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 e Cell viability is poor prior to assay setup resulting in sub optimal results Low level of sample fluorescence e Verify that the System Check procedure was performed and the results passed 15 Related Products Muse M H2A X Activation Dual Detection Kit Catalog No MCH200101 Muse EGFR HRTK Activation Dual Detection Kit Catalog No MCH200102 Muse M PI8K Activation Dual Detection Kit Catalog No MCH200103 Muse MAPK Activation Dual Detection Kit Catalog No MCH200104 Muse Bcl 2 Activation Dual Detection Kit Catalog No MCH2001 05 Muse M Multi Color DNA Damage Kit Catalog No MCH200107 Muse M PI3K MAPK Dual Pathway Activation Kit Catalog No MCH2001 08 Muse RFP LC3 Reporter Autophagy Assay Kit Catalog No MCH2001 10 Muse System Check Kit Catalog No MCH100101 10 Muse Count amp Viability Kit 100T Catalog No MCH100102 11 Muse Annexin V amp Dead Cell Kit Catalog No MCH100105 12 Muse Cell Dispersal Reagent Catalog No MCH100107 pu dE ugue oU gei pue VDS SEX 9 References 1 shvets E et al 2008 Utilizing flow cytometry to monitor autophagy in living mammalian cells Autophagy 4 5 621 8 Zhang L et al 2007 Small molecule regulators of autophagy identified by an image based high throughput screen Proc Natl A
16. n the title bar then Quick Clean from the menu MA pa D Ch Le 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 Results The software performs calculations and displays the data in two plots e A dot plot displaying cells which are chosen for data acquisition and analysis exclusion of cellular debris all performed during adjustment of settings e Ahistogram profile illustrating the fluorescence intensity of the sample acquired All testing sample histogram profiles can be overlaid with the control sample e g no autophagy and compared to monitor the changes in autophagy intensity 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 Sample Number Sample ID Mean Autophagy Intensity Autophagy Induction Ratio Test sample fluorescence versus control sample fluorescence Total number of Cells Statistics Release Mean Autophagy Autophagy Induction esd Intensity Ratio Mean Autophagy Total Sample ID Autophagy Induction of Cells Autophagy i i LC3 detection 198 6 SX 298 995 Select plot to adjust marker gate 988 4 POPUL
17. og No 16466 030 or equivalent e Muse System Check Kit Catalog No MCH100101 e Guava ICF Instrument Cleaning Fluid Catalog No 4200 0140 optional Warnings and Precautions e he 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 components 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 anti LC3 monoclonal antibody may condense within the vial For maximum recovery of the product centrifuge original vial prior to removing cap e Autophagy reagents A and B must be stored in at 2 8 C upon receipt Keep autophagy reagent A lyophilized until ready for use Any leftover reconstituted material can be stored at 20 for up to 6 months avoid repeated freeze thaw e Do not use reagents beyond the expiration date of the kit Storage and Stability All reagents must be stored at 2 8 C All kit components are stable up to six 6 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 pre
18. or use in diagnostic procedures Summary of Protocol Cell Culture and Treatment Add 100 uL 1X Resuspend in 200 uL autophagy reagent 1X Assay Buffer and Culture cells at 40 000 cells per B per tube Incubate Abe eld oh uti Pt well in the 96 well plate overnight for 5 min use Cell Analyzer The next day treat with autophagy E reagent A for 2 6 hours Untreated and Treated m l amp vU Detach cells and transfer to a Muse compatible centrifuge tube and spin at 2500 rpm for 5 min remove Centrifuge cells at 2500 rpm for 5 min and wash with 1X Assay Buffer Materials Provided e 20X Anti LC3 Alexa Fluor amp 555 clone 4E12 Part No CS208164 One vial containing 250 uL CF200097 Autophagy Detection Reagent Pack stored at 2C 8 e Autophagy reagent A Part No CS208212 One vial lyophilized e Autophagy reagent B Part No CS208215 One vial containing 1 mL e 5X Assay Buffer Part No CS202124 One bottle containing 55 mL Materials Required But Not Supplied Reagents Consumables Equipment e Test tubes for sample preparation and e Pipettes with corresponding tips capable of storage accurately measuring 10 1000 uL e Tissue culture reagents i e HBSS PBS e Tabletop centrifuge capable of achieving 300 x w o Ca2 or Mg2 cell dislodging buffers g etc e Mechanical vortex e Deionized Water for buffer dilution e Muse Cell Analyzer e Microcentrifuge tubes with screw caps 1 5 mL VWR Catal
19. paration Time considerations Cell treatment with Autophagy reagent A and selective permeabilization will take approximately 2 hours Cellular staining will take approximately 30 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 Autophagy Reagent A This material is supplied in a lyophilized vial Prior to use reconstitute the contents of the vial in 250 uL deionized water Note It is recommended to aliquot multiple vials and maintain them stored at 20 C Avoid exposure of the reagent to repeated freeze and thaw cycles 2 Autophagy Reagent B Autophagy reagent B is supplied at 10X concentration and should be diluted to 1X with deionized water prior to use Prepared 1X Autophagy Reagent B is stable up to one year Store at 2 8 C 3 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 Assay Instructions General Assay Protocol To monitor autophagosomes 1 Culture cells to approximately 40 000 cells per well in 200 UL growth media in the 96 well plate Incubate overnight in a 37 C incubator with 5 CO 2 The next day for tre
20. ram marker according as described in steps 4 and 6 respectively You cannot adjust the 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 Select to apply changes to other samples Also if you would like to overlay the control and target histograms click the Show Overlay box for a direct visual comparison Lastly the histogram marker can either be visible on the histogram plot or removed To show the histogram marker activate the Show Marker box 2 LC3 INTENSITY Show Overlay Show Marker 13 If no adjustments are needed select Next Run and repeat steps 9 through 11 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 Load sample tube containing DI administrator Your administrator may also have chosen Water to allow you to skip the Quick Clean when the prompt ek appears You can choose to perform additional Quick Cleans at any time during a run by selecting Clean i
21. ram peak as shown in B This marker will isolate the cells of interest and provide a range of detection to record the mean autophagy intensity for both your control and testing samples e As soon as the histogram marker covers a wide range for capture and detection within the histogram plot this will serve as the measure for statistical information as shown in C The marker should be able to enclose all histogram plots both the control and the target samples If this is not feasible it is possible to perform post acquisition adjustments of the histogram marker to capture all required data points A Moving the quadrant marker B Adjusting the histogram marker C Completion 2 2 LC3 INTENSITY LC3 INTENSITY ERI ae 1 Align histogram marker to the ANOPEREY i ON 1 Align histogram marker to the right end 4 right end of the control sample S 2 Adjust sca 3 Uu TA 2 Adjust scale value to capture entire hist entire histogram plot LC3 INTENSITY Next Verify Settings 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 4 POPULATION PROFILE o AUTOPHAGY INDUCTION PROFILE CELL SIZE INDEX e b N I Count 0 1 2 3 o1 2 35 uae LC3 INTENSITY AUTOPHAGY INTENSITY If settings are correct select Next otherwise select Back Back Set Population Pro
22. uid system may be clogged Run a Quick Clean procedure to clean the capillary It can be performed during or after an assay 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 oince autophagy is a constitutive degradation process make sure that Autophagy Reagent A is added 2 hours prior to cell acquisition This reagent functions as a lysosomal degradation inhibitor to prolong the antibody fluorescence detected in autophagosome bound LC3 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 results are obtained A substantial decrease in cell numbers can lead to difficulty in adjusting settings If the concentration of the cell sample is high 21200 cells uL dilute the sample further with Assay Buffer to adjust the cell concentration between 300 and 700 cells uL Although the assay procedure has been optimized to function on the Muse Cell Analyzer at times cell preparation and or health can affect assay performance The wide peaks or false peak may indicate that e The sample is poorly permeabilized 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 d
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