Annexin V FITC Assay Kit

Item No. 600300

 TABLE OF CONTENTS GENERAL INFORMATION
GENERAL INFORMATION 3 Materials Supplied
Materials Supplied
4 Precautions
4 If You Have Problems
Kit will arrive packaged as a 4°C kit. For best results, remove components and store as stated
below.
4 Storage and Stability
4 Materials Needed but Not Supplied Item Item Quantity/Size Storage
Number
INTRODUCTION 5 Background
5 About This Assay 600301 Cell-Based Annexin V FITC 1 vial/50 µl 4°C
PRE-ASSAY PREPARATION 6 Reagent Preparation 600302 Cell-Based Assay Annexin V Binding Buffer (10X) 1 vial/50 ml Room Temperature
ASSAY PROTOCOL 7 Flow Cytometry
10011234 Cell-Based Propidium Iodide Solution 1 vial/250 µl 4°C
8 Fluorescence Microscopy
If any of the items listed above are damaged or missing, please contact our Customer Service
9 Plate Reader Fluorescence Detection
department at (800) 364-9897 or (734) 975-3999. We cannot accept any returns without
prior authorization.
ANALYSIS 10 Performance Characteristics

RESOURCES 12 Troubleshooting
13 References
13 Related Products
! WARNING: This product is for laboratory research use only: not for
administration to humans. Not for human or veterinary diagnostic or
therapeutic use.

14 Warranty and Limitation of Remedy

15 Plate Template
16 Notes

GENERAL INFORMATION 3
Precautions INTRODUCTION
Please read these instructions carefully before beginning this assay.
For research use only. Not for human or diagnostic use. Background
Apoptosis is a programmed process of cell death in which a sequence of events leads to the
If You Have Problems elimination of cells. During an organism’s life cycle, apoptosis plays a vital role in normal
development and maintenance of tissue homeostasis by eliminating old, unnecessary, and
Technical Service Contact Information unhealthy cells. Dysregulation of apoptosis results in pathological conditions including
Phone: 888-526-5351 (USA and Canada only) or 734-975-3888 neurodegenerative diseases, ischemic damage, autoimmune disorders, and cancer.1 The
ability to modulate cell fate (life or death) is thus recognized as an immense therapeutic
Fax: 734-971-3641
potential in drug discovery. Research continues to focus on the elucidation and analysis of
Email: techserv@[Link] signaling pathways that control apoptosis.
Hours: M-F 8:00 AM to 5:30 PM EST One of the hallmarks of the early stages of apoptosis is that membrane phospholipids such
In order for our staff to assist you quickly and efficiently, please be ready to supply the lot as phosphatidylserine and phosphatidylethanolamine redistribute from the inner to outer
number of the kit (found on the outside of the box). leaflet of the membrane bilayer where they are exposed on the cell surface.2,3 Externalization
of phosphatidylserine residues to the outer plasma membrane leaflet allows their detection
via their high-affinity for annexin V, a phospholipid binding protein. Apoptotic cells bound
Storage and Stability with fluorochrome-labeled annexin V can be visualized using fluorescence microscopy,
This kit will perform as specified if stored as directed in the Materials Supplied section and flow cytometry, or a plate reader capable of fluorescence measurements. Compared to
used before the expiration date indicated on the outside of the box. other apoptosis detection methods, such as TdT-mediated dUTP Nick-End Labeling
(TUNEL) analysis, ISEL (in situ end labeling), and DNA laddering analysis for detection
of fragmentation of DNA, Annexin-V analysis has advantages of being more sensitive and
Materials Needed But Not Supplied less time consuming.
1. Adjustable pipettes and a repeat pipettor.
2. 6-, 12-, 24-, or 96-well plates for culturing cells. About This Assay
3. Flow cytometer, fluorescence microscope, or plate reader equipped with laser or filters Cayman’s Annexin V FITC Assay Kit employs a FITC-conjugated annexin V as a probe for
capable of detecting propidium iodide at excitation wavelengths between 520-570 nm phosphatidylserine on the outer membrane of apoptotic cells. Propidium iodide is used as a
and emission wavelengths between 570-610 nm and FITC at excitation/emission = marker of cell death. The reagents provided in the kit are sufficient to run 100 samples when
485/535 nm. using flow cytometry or 500 samples when using a 96-well plate format.
4. A plate centrifuge

4 GENERAL INFORMATION INTRODUCTION 5

 PRE-ASSAY PREPARATION ASSAY PROTOCOL
NOTE: Annexin V FITC is light sensitive. Do not expose to direct intense light. NOTES
• Annexin V FITC and Propidium Iodide are light sensitive. All staining
Reagent Preparation procedures must be performed without direct exposure to intense light.
Incubations should be done in the dark.
Assay Binding Buffer • For all assay protocols described below, it is imperative that samples be
Prepare a working binding buffer by diluting the Cell-Based Assay Annexin V Binding analyzed immediately following completion of the staining.
Buffer (10X) (Item No. 600302) 1:10 in distilled water (for example, add 10 ml of the
Cell-Based Assay Annexin V Binding Buffer (10X) to 90 ml of distilled water). Mix well
and keep at room temperature. The diluted Assay Binding Buffer will be stable for one Flow Cytometry
year at room temperature.
1. Culture cells in 6-, 12-, or 24-well plates at a density of 5 x 105 cells/ml in a CO2
incubator overnight at 37°C. Treat the cells with experimental compounds or vehicle
Annexin V FITC/Propidium Iodide Staining Solution (each sample should be run in duplicate or triplicate). Incubate the cells according to
Prepare an Annexin V FITC/Propidium Iodide Staining Solution by adding 10 µl of Cell- your normal protocol.
Based Annexin V FITC (Item No. 600301) and 10 µl of Cell-Based Propidium Iodide 2. Collect the cells in a test tube and centrifuge at 400 x g for five minutes. Aspirate the
Solution (Item No. 10011234) to 5 ml of the diluted Assay Binding Buffer. Mix well. supernatant.
Prepare this staining solution immediately before adding to the samples. The Annexin
V FITC/Propidium Iodide Staining Solution will be stable for one hour at 4°C. NOTE: 3. Resuspend the cells with 2 ml of diluted Assay Binding Buffer. Mix well to ensure
Protect from light. separation of individual cells.
4. Centrifuge the cells at 400 x g for five minutes and aspirate and discard the
supernatant.
5. Resuspend the cells in 250 µl of Annexin V FITC/Propidium Iodide Staining
Solution. Mix well to ensure separation of individual cells. Incubate the cells in the
dark at room temperature for 10 minutes.
6. Centrifuge the cells at 400 x g for five minutes and aspirate and discard the
supernatant.
7. Resuspend the cells in 2 ml of the diluted Assay Binding Buffer. Mix well to ensure
separation of individual cells.
8. Centrifuge the cells at 400 x g for five minutes and aspirate and discard the
supernatant.
9. Resuspend the cells in 0.5-1 ml of the diluted Assay Binding Buffer.

6 PRE-ASSAY PREPARATION ASSAY PROTOCOL 7

10. Analyze the cells with a flow cytometer. The cells must be analyzed immediately. Dead Plate Reader Fluorescence Detection
cells are stained by propidium iodide and are detectable in the FL2 channel of a flow
cytometer. Early stage apoptotic cells recognized by Annexin V FITC are detectable A 96-well black culture plate should be used for this method. We recommend that the cell
in the FL1 channel. density be 1 x 105-1 x 106 cells/well. Optimal conditions will depend on the cell type.
1. Culture cells in a 96-well black plate at a density of 1 x 105-5 x 105 cells/ml in a CO2
Fluorescence Microscopy incubator overnight at 37°C. Treat the cells with experimental compounds or vehicle
(each sample should be run in duplicate or triplicate). Incubate the cells according to
A 6-, 12-, 24-, or 96-well culture plate can be used for this method. We recommend that your normal protocol.
the cell density be ≤1 x 106 cells/ml. Optimal conditions will depend on the cell type.
2. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
1. Culture cells in 6-, 12-, 24-, or 96-well plates at a density of 5 x 105 cells/ml in a CO2 aspirate and discard supernatant.
incubator overnight at 37°C. Treat the cells with experimental compounds or vehicle
(each sample should be run in duplicate or triplicate). Incubate the cells according to 3. Add 100 µl of diluted Assay Binding Buffer to each well and centrifuge the plate
your normal protocol. for five minutes at 400 x g at room temperature. Carefully aspirate and discard
supernatant.
2. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
aspirate and discard supernatant. 4. Incubate the cells in 50 µl of the Annexin V FITC/Propidium Iodide Staining
Solution for 10 minutes at room temperature.
3. Add 2 mL, 1 ml, 500 µl, or 100 µl of diluted Assay Binding Buffer to each well of a
6-, 12-, 24-, or 96-well plate, respectively. 5. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
aspirate and discard supernatant.
4. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
aspirate and discard supernatant. 6. Add 100 µl of diluted Assay Binding Buffer to each well. The cells are now ready for
analysis with a plate reader equipped with appropriate fluorescence capabilities. Dead
5. Add 800 µl, 400 µl, 200 µl, or 50 µl of Annexin V FITC/Propidium Iodide Staining cells are stained by propidium iodide which display strong fluorescent intensity with
Solution to each well of the 6-, 12-, 24-, or 96-well plate respectively. Incubate for excitation and emission at 560 nm and 595 nm, respectively. Early stage apoptotic
10 minutes at room temperature in the dark. cells stained by Annexin V FITC can be detected with excitation and emission at
6. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully 485 nm and 535 nm, respectively.
aspirate and discard supernatant.
7. Add 2 ml, 1 ml, 500 µl, or 100 µl of diluted Assay Binding Buffer to each well of a
6-, 12-, 24-, or 96-well plate respectively.
8. Repeat steps 6-7.
9. Examine the cells by fluorescence microscopy. Cells must be analyzed immediately.
Dead cells are stained by propidium iodide and can be detected using a filter designed
to detect rhodamine (excitation/emission = 540/570 nm) or Texas Red (excitation/
emission = 590/610 nm). Early stage apoptotic cells stained by Annexin V FITC
can be detected using a filter designed to detect fluorescein (excitation/emission =
485/535).

8 ASSAY PROTOCOL ASSAY PROTOCOL 9

 ANALYSIS
Performance Characteristics

Figure 2. Staurosporine induces apoptosis in Jurkat cells as measured by flow

cytometry. Jurkat cells were plated at a density of 1 x 106 cells/ml in a 6-well plate.
The next day, cells were treated with vehicle (control), 0.5 µg/ml staurosporine for
four hours, or 0.5 µg/ml staurosporine overnight in a CO2 incubator at 37°C. Cells were
then processed for staining of dead cells and apoptotic cells. A majority of control
Staurosporine
Figure 1. Staurosporine induces
inducesapoptosis
apoptosis in in RAWRAW 264.7264.7cells,cells, as
as measured cells (left panel) were not stained with either Annexin V FITC or propidium iodide and
by anmeasured
increase of byAnnexin
an increase
V FITCof Annexin
positive V FITC
cells. RAW 264.7positive [Link]
cells were RAWat a appear in lower left quadrant. When cells were treated with 0.5 µg/ml staurosporine
density
264.7 x 104were
of 5cells cells/well
plated in at
a 96-well
a densityplate.
of The 104 day,
5 xnext cells were
cells/well in a treated
96-wellwith for four hours (middle panel), there is a significant increase in Annexin V FITC positive
vehicle
plate.(control)
The next or 8day,
µg/ml
cellsofwere
staurosporine
treated with (treatment) for five hours
vehicle (control) in a CO2
or 8 µg/ml cells (lower right quadrant) whereas there is no change in cells that were both
incubator at 37°C. Cells(treatment)
of staurosporine were then processed
for five hoursfor staining
in a COof2 dead cells and
incubator Annexin
at 37°C. propidium iodide and Annexin V FITC positive (upper right quadrant). In contrast,
V FITC
Cells were then processed for staining of dead cells and Annexin aV few
according to the protocol above. Left Panel: in control cells, there are cells treated with 0.5 µg/ml staurosporine overnight (right panel) had a significant
propidium iodide positive increase in both Annexin V FITC positive cells (lower right quadrant) and cells that
FITC according to thedead cells above.
protocol (arrow heads) and few
Left Panel: of these
in control cellsthere
cells, are from
were both propidium iodide and Annexin V FITC positive (upper right quadrant). The
apoptosis (thick arrow, both propidium iodide and Annexin V FITC positive). A thin
arroware a few propidium
indicates iodide
a cell at an early positivestage,
apoptotic deadwhichcells is(arrow
Annexin heads)
V FITCand few but
positive results indicate that staurosporine causes Jurkat cell death through apoptosis and
of these cells are from apoptosis (thick arrow, both propidium
propidium iodide negative. Right Panel: cells treated with 8 µg/ml of staurosporine, iodide the effect can be detected as early as four hours after the treatment.
show and AnnexininVboth
an increase FITC positive).iodide
propidium A thin arrow cells
positive indicates a cell atAnnexin
(arrowheads), an earlyV FITC
apoptotic
positive stage,
cells (thin which
arrows), andis cells
Annexin
which V are
FITC positive
both propidium but propidium
iodide and iodide
Annexin V
FITCnegative. Right
positive (thick Panel:indicating
arrows), cells treated with 8 of
that treatment µg/ml of staurosporine,
staurosporine in RAW 264.7
cellsshow
causes apoptosis.
an increase in both propidium iodide positive cells
(arrowheads), Annexin V FITC positive cells (thin arrows), and cells
which are both propidium iodide and Annexin V FITC positive (thick
arrows), indicating that treatment of staurosporine in RAW 264.7 cells
causes apoptosis.

10 ANALYSIS ANALYSIS 11
 RESOURCES Reference
1. Schiller, M., Bekerdjian-Ding, I., Heyder, P., et al. Autoantigens are translocated into
Troubleshooting small apoptotic bodies during early stages of apoptosis. Cell Death and Differentiation
15, 183-191 (2008).
2. van Engeland, M., Nieland, L.J.W., Ramaekers, F.C.S., et al. Annexin V-affinity assay:
Problem Possible Causes Recommended Solutions A review on an apoptosis detection system based on phosphatidylserine exposure.
Cytometry 31, 1-9 (1998).
Strong staining for both Annexin Cells are not healthy A. Use only healthy cells 3. Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35(4), 495-
V FITC and propidium iodide in B. Keep staining time to 10 minutes 516 (2007).
all samples, including controls (prolonged incubation time increases
cell death) Related Products
High level of Annexin V FITC Cells are damaged during Process the sample gently; for example, 7-AAD Cell Viability Assay Kit - Item No. 10009856
staining in all samples, including harvesting or processing for disperse cells gently by pipetting cells up 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit - Item No. 600120
controls staining and down; Do not vortex the cells; Do not Apoptotic Blebs Assay Kit - Item No. 10010750
use scraping for adherent cells Caspase-3 Fluorescence Assay Kit - Item No. 10009135
Glutathione Cell-Based Detection Kit (Blue Fluorescence) - Item No. 600360
No signal for Annexin V FITC A. Annexin V FITC/Propidium A. Use right amount of Annexin V FITC Glycolysis Cell-Based Assay Kit - Item No. 600450
Iodide Staining Solution to prepare Annexin V FITC/Propidium JC-1 Mitochondrial Membrane Potential Assay Kit - Item No. 10009172
not prepared properly Iodide Staining Solution
B. Cells lost during B. Decrease treatment time or LDH Cytotoxicity Assay Kit - Item No. 10008882
processing compound dosage MTT Cell Proliferation Assay Kit - Item No. 10009365
Multi-Drug Resistance Assay Kit (Calcein AM) - Item No. 600370
Multi-Parameter Apoptosis Assay Kit - Item No. 600330
NAD+/NADH Cell-Based Assay Kit - Item No. 600480
Neutrophil Elastase Activity Assay Kit - Item No. 600610
20S Proteasome Assay Kit - Item No. 10008041
WST-1 Cell Proliferation Assay Kit - Item No. 10008883
WST-8 Cell Proliferation Assay Kit - Item No. 10010199
XTT Cell Proliferation Assay Kit - Item No. 10010200

12 RESOURCES RESOURCES 13
Warranty and Limitation of Remedy

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direct, indirect, incidental or consequential damages, even if Cayman is informed about
their possible existence. This limitation of liability does not apply in the case of intentional
acts or negligence of Cayman, its directors or its employees.
Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a refund
of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer, of all
material that does not meet our specifications.
Said refund or replacement is conditioned on Buyer giving written notice to Cayman
within thirty (30) days after arrival of the material at its destination. Failure of Buyer to
give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims
hereunder with respect to said material.
For further details, please refer to our Warranty and Limitation of Remedy located on
our website and in our catalog.

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 NOTES

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or machine-readable form without prior consent, in writing, from Cayman Chemical
Company.
©02/11/2013, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed
in U.S.A.
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