Muse® Annexin V & Dead Cell Kit

Technical Support
Telephone: 512-381-4397
North America Toll Free: 1-877-785-2323
International Toll Free: + 800-2939-4959
Email: support@luminexcorp.com
www.luminexcorp.com

For Research Use Only. Not for use in diagnostic Luminex Corporation
procedures.
4600-3384, Rev F 12212 Technology Blvd.
Catalog No. MCH100105 (100 tests) Austin, TX 78727
November 2019 U.S.A.
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Muse Annexin V & Dead Cell Kit

Application
The Muse® Annexin V & Dead Cell Assay allows for the quantitative analysis of live, early and late apoptosis, and
cell death on both adherent and suspension cell lines on the Guava® Muse® Cell Analyzer. Minimal sample prepara-
tion is required in this no-wash, mix-and-read assay to obtain accurate and precise results. The software provides:

• Concentrations (cells/mL) for live, early apoptotic, late apoptotic, total apoptotic, and dead cells,
• Percentage of live, early apoptotic, late apoptotic, total apoptotic, and dead cells
The Muse Annexin V & Dead Cell Assay is for use with the Guava Muse Cell Analyzer. The Muse System makes
sophisticated fluorescent-based analysis fast, easy, convenient, and affordable. Sample preparation is minimal, and
after loading samples onto Guava Muse Cell Analyzer, intuitive software provides detailed or summary analysis of
your cell sample in a few short steps.

Sufficient reagent is provided for the preparation and analysis of 100 tests.

Test Principle
Apoptosis, or programmed cell death, is an important and
active regulatory pathway of cell growth and proliferation.
Cells respond to specific induction signals by initiating
intracellular processes that result in characteristic physio-
logical changes. Among these are externalization of phos-
phatidylserine (PS) to the cell surface, cleavage and
degradation of specific cellular proteins, compaction and PS molecules translocate to the outer surface of the cell
fragmentation of nuclear chromatin, and loss of membrane membrane where Annexin V can readily bind them.
integrity (in late stages).1-5 Annexin V is a calcium-depen-
dent phospholipid-binding protein with a high affinity for PS, a membrane component normally localized to the
internal face of the cell membrane.6-8 Early in the apoptotic pathway, molecules of PS are translocated to the outer
surface of the cell membrane where Annexin V can readily bind them.9-14

The Muse® Annexin V & Dead Cell Assay utilizes Annexin V to detect PS on the external membrane of apoptotic
cells. A dead cell marker is also used as an indicator of cell membrane structural integrity.15 It is excluded from live,
healthy cells, as well as early apoptotic cells. Four populations of cells can be distinguished in this assay:

• non-apoptotic cells: Annexin V (-) and 7-AAD (-)

• early apoptotic cells: Annexin V (+) and 7-AAD (-)
• late stage apoptotic and dead cells: Annexin V (+) and 7-AAD (+)
• mostly nuclear debris: Annexin V (-) and 7-AAD (+)

} Cells Quadrant
1 (UL) - dead cells
2 (UR) - late apoptotic/dead cells
3 (LL) - live cells

}
4 (LR) - early apoptotic cells
Debris

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Each plot has moveable markers. The first plot has a threshold marker, allowing you to eliminate debris based on
size, as well as a gate, allowing you to gate on cells. The second plot has quadrant markers, allowing you to obtain
statistics on four cell populations—live, early apoptotic, late apoptotic, and dead.

Summary of Protocol
Add 100 μL of Muse® Add 100 μL of cells**
Annexin V & Dead Cell in suspension to
Reagent to each tube. each tube.
Incubate for
Prepare cellular samples for 20 minutes at
Culture cells, including for incubation with the Muse® room temperature.
positive and negative Annexin V & Dead Cell reagent.
controls, for appropriate
time to induce apoptosis.

** Adherent cells have been validated for this assay. For information on preparing adherent cells, see Appendix A: Cell
Sample Preparation on page 14.

Kit Components
Muse® Annexin V & Dead Cell Reagent (Part No. 4700-1485, 100 tests/bottle)

Materials Required but Not Supplied

• Guava® Muse® Cell Analyzer
• Cell suspensions untreated and treated to undergo apoptosis
• Micropipettors
• Disposable micropipettor tips
• Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Catalog No. 16466-030, or equivalent)
• Vortex mixer
• Disposable gloves
• 20% bleach solution
• Deionized water
• Centrifuge
• Muse® Cell Dispersal Reagent (Catalog No. MCH100107), optional
• Guava® ICF Instrument Cleaning Fluid (Catalog No. 4200-0140), optional
• Muse® System Check Kit (Catalog No. MCH100101), optional

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Precautions
• The Muse® Annexin V & Dead Cell Reagent is intended for research use only.
• Wear proper laboratory attire (lab coat, gloves, safety glasses) when handling this reagent.
• The Muse Annexin V & Dead Cell Reagent contains dyes that may be carcinogenic and/or mutagenic.
Exercise standard precautions when obtaining, handling, and disposing of potentially carcinogenic and
mutagenic reagents. Refer to the SDS for specific information on hazardous materials.
• The Annexin V & Dead Cell Reagent contains sodium azide, which is toxic if ingested. Reagents containing
sodium azide should be considered a poison. If products containing sodium azide are swallowed, seek
medical advice immediately and show product container or label. (Refer to NIOSH, National Institute for
Occupational Safety and Health; CAS#: 2628-22-8; and also to GHS, The Globally Harmonized System of
Classification and Labeling of Chemicals.) Aqueous solutions of sodium azide, when mixed with acids,
may liberate toxic gas. Any reagents containing sodium azide should be evaluated for proper disposal.
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Upon dis-
posal, flush with large volumes of water to prevent build-up in plumbing. Check with regulatory agencies
to determine at what concentration sodium azide may cause a product to be regulated as hazardous.
• Avoid microbial contamination of the solution, which may cause erroneous results.
• All biological specimens and materials should be handled as if capable of transmitting infection and dis-
posed of with proper precautions in accordance with federal, state, and local regulations. Never pipette by
mouth. Avoid specimen contact with skin and mucous membranes.
• Exercise care to avoid cross contamination of samples during all steps of this procedure, as this may lead
to erroneous results.
• The fluorescent dyes in this reagent are light sensitive. Store in the dark and shield from excessive expo-
sure to light.
• 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.
• During storage and shipment, small volumes of product will occasionally become entrapped in the seal of
the product vial. For maximum recovery of the product, centrifuge the vial briefly prior to removing the cap.
• Do not use the reagent beyond the expiration date.
• Safety Data Sheets (SDSs) for kit reagents are available from our website (www.luminexcorp.com) or by
contacting Luminex Technical Support.

Storage
• Store the Muse® Annexin V & Dead Cell Reagent refrigerated at 2 to 8°C. Do not freeze. Refer to the expi-
ration date on the package label. Do not use the reagent after the expiration date.
• The Muse Annexin V & Dead Cell Reagent contains light-sensitive dyes. Shield from excessive exposure to
light.

Before You Begin

This protocol was developed to allow direct determination of the percent of early and late apoptotic populations
induced in cultures. For optimal throughput, final cell concentrations should be between 2 x 104 and 1 x 105 cells/
well (or 1 x 105 to 5 x 105 cells/mL), although apoptosis can be detected in cultures with as few as 2 x 103 cells/
well (or 1 x 104 cells/mL). Care should be taken to keep cell concentrations as constant as possible in all samples
of an experiment. The mean fluorescent intensity of Annexin V bound to early and late apoptotic cells can vary

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significantly with a two-fold change in cell concentration, although the percentage of cells bound by Annexin V
remains constant. However, if the cell concentration exceeds 5 x 105 cells/mL, the Annexin V reagent may be in
limiting concentration and will therefore bind to fewer cells, resulting in lower percentages for both early and late
apoptotic cells.

Cells should be acquired shortly after the sample preparation is completed. While some cell lines have been
shown to yield stable results for up to 3 hours, others are stable for only 1 hour. This time variability is a conse-
quence of using live, unfixed cells. You should determine the stability of results for your own cells. We strongly dis-
courage fixing the cells after sample preparation to enhance stability, as the fixation will permeabilize all cells
increasing the percentage of cells stained with the dead cell marker, and resulting in an underestimation of the
early apoptotic cells and an overestimation of the late apoptotic and dead cells.

The following procedures for cell staining are guidelines. Different cell types have varying phosphatidylserine (PS)
content in their cell membranes.16-18 Upon induction of apoptosis, different cell types vary in the amount of PS
exposed on the cell surface.11,19 You may need to adjust the amount of Muse® Annexin V & Dead Cell Reagent
used for optimal staining of your cell samples. If this is the case, please follow the recommendations described in
Cell Staining Procedure.

Time considerations: Staining cells with the Muse Annexin V & Dead Cell Reagent takes 20 minutes. Acquiring
data on the Guava® Muse Cell Analyzer takes approximately 2 minutes per sample. However, preparing cells for
testing requires periodic maintenance and cultivation several days in advance. Once you cultivate the proper num-
ber of cells for your experiment, it takes an additional 2 to 48 hours of culture with various inducers to stimulate
detectable apoptosis.

For details on how to culture and prepare cell samples, including positive and negative control samples, see Appen-
dix A: Cell Sample Preparation on page 14.

Always run a System Check prior to performing the assay. For details refer to the Guava Muse Cell Analyzer User’s
Guide.

Staining Protocol
1. Allow the Muse® Annexin V & Dead Cell Reagent to warm to room temperature.
2. Add 100 µL of cells in suspension to each tube. For instructions on preparing cell suspensions, see Appendix A:
Cell Sample Preparation on page 14.

3. Add 100 µL of the Muse Annexin V & Dead Cell Reagent to each tube.

NOTE: Should your induced cells express large amounts of PS, it may be necessary to add more reagent.
You can add up to 150 µL of the Annexin V & Dead Cell Reagent to each tube. If you need to use
more reagent for optimal staining, then it is better to decrease the volume of medium that the cells
are in from 100 to 50 µL and add between 150 to 175 µL (up to 200 µL) of the reagent.
4. Mix thoroughly by pipetting up and down or vortexing at a medium speed for 3 to 5 seconds.
5. Stain samples for 20 minutes at room temperature in the dark.

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Setup and Acquisition on the Guava® Muse® Cell

Analyzer
Run a System Check prior to performing the assay. For information on Muse® System Check, refer to the Guava®
Muse Cell Analyzer User’s Guide.

1. Select Annexin V & Dead Cell from the main menu.

2. Select Run Assay.

3. Adjust the instrument settings.

• Load the sample for adjusting the settings and select Run.
NOTE: Perform the adjust settings step using a negative control,
then verify the settings using a positive control.
• Or, to retrieve previously saved instrument settings, select Retrieve
Settings. For more information on retrieving settings, see the Guava
Muse Cell Analyzer User’s Guide.
4. Fine tune the settings for the ANNEXIN vs. CELL SIZE INDEX plot, if
necessary.
• Adjust the CELL SIZE INDEX slider to the left of the plot and the
ANNEXIN V slider below the plot to move the cellular populations
into the green region.

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• Drag the threshold up or down to exclude 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.
• Touch the upper-left and/or lower-right corners of the gate and drag to adjust the gate size.

NOTE: If the acquisition times out (after 2 minutes), you can select Back to restart the adjust settings step
or Next to accept the settings and continue to the next step.

Touch threshold to Touch and drag

move up/down. upper-left or lower-
right corner to
adjust the gate.

These examples
show typical gate
and threshold
settings.

Negative control sample Positive control sample

5. Select Next when you’ve completed the adjustments.

6. Fine tune the settings for the ANNEXIN V vs. VIABILITY plot, if necessary.
• Adjust the VIABILITY slider to place all populations (live, dead, and apoptotic) on scale.
• 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. To

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adjust the angle of either line, touch the solid circle and drag in an arc, or touch the arrow buttons below
the plot.

Touch center Touch a solid circle on

circle to move the either line (horizontal
fixed quadrant or vertical) to adjust
markers in any the angle of the line.
direction.

Negative control sample Positive control sample

7. Select Next when the adjustments are complete.

8. Verify the settings for the negative control. Then select Back and repeat steps 4 through 7 using a positive
control. When the settings are correct, select Next.

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9. Enter the sample ID by touching the field, then using the keypad to input the ID. Touch Done when you’ve fin-
ished entering the ID. If necessary, change the Events to Acquire and/or Dilution Factor by touching the field,
then selecting the value from the pop-up menu. Select Next.

10. Mix the first sample and load it on the instrument. Select Run to run the sample.

11. When acquisition is complete, the results are displayed. If necessary, select Plots to display dot plots for the
sample.

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You can view or change the sample ID and dilution factor, 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.

Select to
hide plots.

Select to
display plots.

12. (Optional) If changes are needed to the gate or markers, touch a plot to enlarge it, then adjust the cell size
gate or markers, 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 or marker 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.

Apply changes Select to apply

changes to other
samples.

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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 appears. 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 Guava Muse Cell Analyzer User’s Guide for more information.

Results
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.

Events in each of the four quadrants are as follows:

• sample number
• sample ID
• concentration (cells/mL) of cells in each quadrant
lower-left: viable cells, not undergoing detectable apoptosis [Annexin V-PE (–) and dead cell marker (–)]
lower-right: cells in the early stages of apoptosis [Annexin V-PE (+) and dead cell marker (–)]
upper-right: cells in the late stages of apoptosis or dead by apoptotic mechanisms [Annexin V-PE (+) and
dead cell marker (+)]
upper-left: cells that have died via necrosis but not through the apoptotic pathway [Annexin V-PE (–) and
dead cell marker (+)]
• percentage of gated cells in each quadrant
• concentration and percentage of total apoptotic cells (cells in upper-right and lower-right quadrants)
• dilution factor (input value)
• fluorescence intensity values for live and apoptotic populations
In Figures A and B, healthy Jurkat cells were treated with staurosporine to induce apoptosis, then stained with the
Muse® Annexin V & Dead Cell Reagent, and acquired on the Guava® Muse® Cell Analyzer. Figure A shows sum-
mary data, while Figure B shows results displayed with optional dot plots. The statistics show the concentration
(cells/mL) for the events in each quadrant and the percentage of gated cells in each quadrant, as well as the con-
centration and percentage of total apoptotic cells. The first plot in Figure B shows Annexin V vs. Cell Size and the
second plot shows Annexin V vs. Viability.

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Figures A and B.
A B

Technical Tips
1. 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.
2. Multiple acquisitions of a cell sample minimizes sampling error. Statistically, multiple acquisitions yield more
accurate cell counts and viability results.
3. The default number of events to acquire is 2000. You may select a different number; however, your statistical
error will increase as you decrease the number of acquisition events.
4. If results deviate from expected values, prepare a freshly stained sample and reacquire the data.
5. If the cell count results deviate from expected values, check that the correct values were entered for dilution fac-
tor of the cell suspension. The Annexin V & Dead Cell application can be used to recalculate cell counts. Open
the data file corresponding to the mistaken entry. Reenter the correct dilution factor and the cell count values will
be recalculated automatically.
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. 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 µL of sample in the tube. If not, add additional buffer to bring the volume up to 100 µL or proceed to the
next sample. If the sample volume is greater than 100 µL, 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 Back-
flush 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 Guava® Muse® Cell Analyzer User’s Guide for addi-
tional troubleshooting tips, or contact Luminex Technical Support for help.
8. Annexin V & Dead Cell works best with samples in a homogeneous, single cell suspension. Cell aggregates
may clog or be excluded from the flow cell, affecting the accuracy of your results. If you want to use the Muse
Annexin V & Dead Cell assay with a “clumpy” cell line, such as Chinese Hamster Ovary (CHO) cells, we rec-
ommend that you order Muse Cell Dispersal Reagent (Catalog No. MCH100107) to disaggregate the cells.
Contact Customer Service or visit our website at www.luminexcorp.com for detailed information on the Muse
Cell Dispersal Reagent and assay method. For more troubleshooting tips, refer to the Guava Muse Cell Analyzer
User’s Guide.

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Troubleshooting

Potential Problem Experimental Suggestion

Acquisition taking longer than Ensure that the System Check procedure was run and passed. If the progress
expected or progress bar bar stops during acquisition, the fluid system may be clogged. Run a Quick
stops during acquisition Clean procedure.

Instrument clogging Run a Quick Clean to clean out capillary. This procedure can be performed
Too many cells during or after an assay. This will wash away any material forming within the
glass capillary walls.

Low Cell Concentration warn- The sample concentration may be too low. The assay instructions are opti-
ing during acquisition mized to give you a range of cells between 100–500 cells/µL in the final sam-
ple volume so accurate population count results are obtained. Repeat sample
preparation with a lower dilution factor to allow for adequate cell numbers. A
substantial decrease in cell numbers can lead to difficulty in adjusting settings.

High Cell Concentration If the concentration of the stained cell sample for acquisition is high (>500
warning during acquisition cells/µL), the accuracy of data will most likely be compromised. Dilute the
sample further with 1X Assay Buffer HSC to adjust the cell concentration
below 500 cells/µL. For best results, it is recommended that the cell concen-
tration is in the range of 200 to 300 cells/µL.

Background staining and/or If all samples appear to be induced even when low levels of induction are
non-specific staining of cells expected, your cultures may be compromised. It is important to run negative
control samples for each experiment. The negative control should be a sample
from your cell culture, not treated to induce apoptosis. Typically, negative con-
trol samples show a low level of Annexin V-PE and/or dead cell marker posi-
tive cells that are distinct from that of induced cells, because healthy cell
cultures contain a small number of apoptotic and/or dead cells. However, sub-
optimal culture conditions may stress cells in culture, causing them to
undergo apoptosis in the absence of experimental induction treatment. The
negative control from a stressed culture often shows increased Annexin V
and/or dead cell marker reactivity.

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Potential Problem Experimental Suggestion

Low level of staining • Although the assay procedure has been optimized to function utilizing
multiple cell types, every cell line behaves differently. A lack of signal may
indicate that excess dilution factors may need to be altered to obtain
accurate results.
• If there are low levels of Annexin V-PE staining, it is possible that your
cells may not be fully induced. Translocation of phosphatidylserine (PS) to
the cell surface is an early event in apoptosis, which can precede DNA
fragmentation by several hours, and can be reversed in some cases.20 The
Annexin V-PE staining results can vary over time as apoptosis progresses.
To determine optimal apoptotic induction, conduct a time-course study to
achieve the best results for Annexin V-PE staining.
• If there are no Annexin V-positive cells, your cells may not have induced
or the Annexin V-PE may have not been taken up correctly by the cells.
Positive control samples are recommended for each experiment. Positive
controls should be appropriate for comparison with the test procedure or
test cell population. Use a cell line previously characterized as inducible
for apoptosis. Treatments used to induce apoptosis in various cell lines
include a) serum starvation, b) activation of cell surface receptors such as
Fas, TNFR1, or TCR, c) UV irradiation, and d) treatment with a compound
known to induce apoptosis in your cell line.

Poor separation of live and If the separation between populations is poor, the Annexin V concentration
apoptotic populations may be too low. Muse® Annexin V & Dead Cell reagent has been formulated
for optimal performance using Jurkat, CHO, HeLa, PC3, HB, and Daudi cells.
Other cells may show different patterns of reactivity that require adjustments
to the amount of reagent used. For best results, titer the Annexin V & Dead
Cell reagent to determine the amount for maximal staining of cells.

Precipitate forms during incu- Repeat the experiment with freshly induced cells, and increase the percentage
bation of the cells with the of BSA, FBS, or NHS used during the staining procedure.
Annexin V & Dead Cell
Reagent

Variability in day-to-day • If the results are inconsistent, check that the samples were well mixed prior
experiments to acquisition. Cells may quickly settle in your samples and your results will
be inaccurate unless the cells are mixed just prior to acquisition.
• Monitor experimental cell cultures to ensure that cell viability and cell
numbers being analyzed are consistent. Any drop in cell numbers or via-
bility can influence experimental results.
• If there appears to be day-to-day variation of the staining pattern, ensure
the Guava® Muse® Cell Analyzer is working properly. Run the Muse Sys-
tem Check Procedure (Part No. MCH100101) to verify proper instrument
function and accuracy.

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Limitations
• The results of the assay are dependent upon proper handling of samples, reagents, and instruments.
• Cell types vary in the PS content of their cell membrane.16-18 The amount of PS exposed on the cell surface
varies among cell types after apoptosis is induced.11,19 This assay does not detect early apoptosis in cell
types that do not translocate PS to the cell surface upon induction of apoptosis.
• The Muse® Annexin V & Dead Cell Reagent is designed for use on unfixed cells. Fixing cells yields inaccu-
rate results.
• The Guava® Muse Cell Analyzer and Muse® Annexin V & Dead Cell Reagent yield optimal results when
the stained cell sample used for acquisition is between 1 x 104 to 5 x 105 cells/mL. To obtain the most
accurate results, adjust the cell concentrations to within the recommended range. However, to optimize
throughput, Luminex recommends using between 1 x 105 to 5 x 105 cells/mL when possible.

Appendix A: Cell Sample Preparation

Preparing Non-Adherent and Adherent Cells
The following protocols describe how to harvest non-adherent or adherent cells cultured in 96-well plates, as well
as non-adherent or adherent cells cultured in flasks or other tissue culture vessels. Each of the culturing conditions
requires different protocols to harvest the cells.

Preparing non-adherent cells

1. Set up initial culture conditions, such that after culture and treatment, cells are at a concentration of 1 x 105 to
1 x 107 cells/mL in low serum- or albumin containing medium.
2. Proceed to Staining Protocol on page 4.

Preparing adherent cells

For harvesting adherent cells, use your method of removal. Reagents such as EDTA or trypsin can be used to disso-
ciate the cells from the flask and should create single-cell suspensions. If using mechanical means to dislodge the
cells, additional reagents such as Muse® Cell Dispersal Reagent (Catalog No. MCH100107) may be used to disso-
ciate clumps.

1. Using your preferred method for dissociation, detach the cells from their culture vessel.
2. Add fresh serum- or albumin-containing medium to each well so the final concentration is between 1 x 105 to
1 x 107cells/mL.
3. Proceed to Staining Protocol on page 4.

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References
1. Kerr JFR, et al. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br
J Cancer. 1972;26:239-257.
2. Wyllie AH, et al. Cell Death: The significance of apoptosis. Int Rev Cytol. 1980;68:251-306.
3. Wyllie AH. Apoptosis. Br J Cancer. 1993;67:205-208.
4. Majno G, Joris I. Apoptosis, oncosis, and necrosis: An overview of cell death. Am J Pathol. 1995;146:3-15.
5. Rudin CM, Thompson CB. Apoptosis and disease: Regulation and clinical relevance of programmed cell death.
Ann Rev Med. 1997;48:267-281.
6. Tait JF, et al. Phospholipid binding properties of human placental anticoagulant protein-I, a member of the
lipocortin family. J Biol Chem. 1989;264:7944-7949.
7. Andree HAM, et al. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol
Chem. 1990;265:4923-4928.
8. van Heerde WL, et al. The complexity of the phospholipid binding protein Annexin V. Thrombosis and Haemo-
stasis. 1995;73:172-179.
9. Fadok VA, et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific rec-
ognition and removal by macrophages. J Immunol. 1992;148:2207-2216.
10. Koopman G, et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells under-
going apoptosis. Blood. 1994;84:1415-1420.
11. Martin SJ, et al. Early redistribution of plasma membrane phosphatidylserine in a general feature of apoptosis
regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182:1545-
1556.
12. Vermes I, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on
early apoptotic cells using fluorescein labelled Annexin V. J Immunol Meth. 1995;184:39-51.
13. van Engeland M, et al. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of
adherent cells in culture. Cytometry. 1996;24:131-139.
14. van den Eijnde SM, et al. In situ detection of apoptosis during embryogenesis with Annexin V: From whole
mount to ultrastructure. Cytometry. 1997;29:313-320.
15. Schmidt I, et al. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immu-
nofluorescence in single laser flow cytometry. Cytometry. 1992;13:204-208.
16. Op den Kamp JAF. Lipid asymmetry in membranes. Ann Rev Biochem. 1979;48:47-71.
17. Zachowski A. Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem
J. 1993;294:1-14.
18. Tait JF, Gibson D. Measurement of membrane phospholipid asymmetry in normal and sickle-cell erythrocytes
by emans of annexin V binding. J Lab Clin Med. 1994;123:741-748.
19. Frey T. Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some
changes in some model systems. Cytometry. 1997;28:253-263.
20. Hammill AK, et al. Annexin V staining due to loss of membrane asymmetry can be reversible and precede
commitment to apoptotic death. Exp Cell Res. 1999;251:16-21.

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Related Kits
• Muse® System Check Kit – MCH100101
• Muse® Count & Viability Kit (100T) – MCH100102
• Muse® Count & Viability Kit (600T) – MCH600103
• Muse® Count & Viability Kit (200X) – MCH100104
• Muse® Cell Cycle Kit – MCH100106
• Muse® Cell Dispersal Reagent – MCH100107

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