Annexin V-PE/7-AAD Apoptosis Detection Kit

20T, 50T, 100T

Flow cytometry; fluorescence microscopy

Annexin V-PE / 7-AAD double staining

Annexin V-PE and 7-AAD: 4 °C, dark. Binding Buffer (4×): 4 °C.

Phosphatidylserine (PS) is a negatively charged phospholipid. In normal cells, PS is distributed exclusively on the inner leaflet of the plasma membrane lipid bilayer, whereas in early apoptosis, membrane PS is flipped from the inner to the outer leaflet of the lipid membrane and becomes exposed on the outer surface of the cell membrane. Annexin V is a Ca²⁺-dependent phospholipid-binding protein with a molecular weight of 35–36 kDa. Owing to its high affinity for phosphatidylserine, it binds to the plasma membrane of early apoptotic cells through the phosphatidylserine exposed on the outer cell surface. Annexin V is therefore widely recognized as one of the most sensitive markers for detecting early apoptosis.

Annexin V labeled with the fluorescent protein PE can be used as a probe to detect the occurrence of apoptosis by fluorescence microscopy or flow cytometry. 7-AAD (7-amino-actinomycin D) is a nucleic acid dye that cannot penetrate the intact plasma membrane of normal or early apoptotic cells; however, in mid-to-late apoptotic and necrotic cells, 7-AAD can penetrate the cell membrane and stain the nucleus red. Double staining with Annexin V-PE and 7-AAD therefore allows cells at different stages of apoptosis to be distinguished.

1. Micropipettes

2. PBS

3. Trypsin digestion solution (EDTA-free)

4. Low-speed centrifuge

5. Flow cytometer

1. Preparation of cell samples

a) Suspension cells:

i. Collect the cells into a centrifuge tube and centrifuge at 1000–2000 rpm for 5 min; carefully remove the supernatant.

ii. Gently resuspend the cells in 1 ml of PBS pre-chilled to 4 °C, count the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

iii. Add another 1 ml of PBS pre-chilled to 4 °C to resuspend the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

b) Adherent cells:

i. Transfer the culture medium to a centrifuge tube, wash the adherent cells once with PBS, and add an appropriate volume of EDTA-free trypsin cell digestion solution to detach the cells.

ii. Incubate at room temperature until the cells can be detached by gentle pipetting, then aspirate the trypsin digestion solution. Avoid over-digestion with trypsin.

iii. Add the culture medium collected in step i, mix briefly, transfer to a centrifuge tube, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

Note: Adding the culture medium from step i serves, on the one hand, to collect cells that have already become suspended owing to apoptosis or necrosis; on the other hand, the serum in the culture medium effectively inhibits or neutralizes residual trypsin — residual trypsin would digest and degrade the Annexin V conjugate added subsequently and cause staining failure.

iv. Gently resuspend the cells in 1 ml of PBS pre-chilled to 4 °C, count the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

v. Add another 1 ml of PBS pre-chilled to 4 °C to resuspend the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

2. Dilute the Binding Buffer 1:4 with deionized water (4 ml of Binding Buffer (4×) + 12 ml of deionized water).

3. Resuspend the cells in 250 µl of Binding Buffer and adjust the concentration to 1 × 10⁶/ml.

4. Transfer 100 µl of the cell suspension to a 5 ml flow cytometry tube and add 5 µl of Annexin V-PE; mix gently.

5. Incubate for 5 min at room temperature (20–25 °C) protected from light.

6. Add 10 µl of 7-AAD solution 5 min before analysis and mix gently.

7. Immediately before analysis, add 400 µl of PBS to the reaction tube to resuspend the cells, keep protected from light, and analyze promptly by flow cytometry (FC).

1. This kit is for research use only.

2. For small-volume reagents, centrifuge for a few seconds to collect the reagent at the bottom of the tube before opening the cap.

3. 7-AAD (7-amino-actinomycin D) is toxic. Wear gloves when handling it and avoid contact with skin, eyes, and mucous membranes.

4. Annexin V-PE contains the toxic component sodium azide (NaN₃). Wear gloves when handling it and avoid contact with skin, eyes, and mucous membranes.

5. This kit is intended for the detection of viable cells; when analyzed by flow cytometry, the number of cells should not be less than 1 × 10⁶.

6. Samples should be analyzed as soon as possible after staining; prolonged standing may increase the number of apoptotic or necrotic cells.

7. The Annexin V assay is suitable for cells grown in suspension, such as lymphocytes. For adherent cells, trypsin and other digestive treatments damage the cell membrane and cause a high rate of false positives, while scraping with a cell scraper causes the cells to clump together and interferes with detection; the trypsin-detached cells may be kept in PBS containing 2% BSA to prevent further damage. Although some laboratories in China and abroad currently use this method to detect adherent cells, we do not recommend it, because its reproducibility is poor and great care is required in handling.

8. Residual trypsin from digesting adherent cells will digest and degrade Annexin V-PE and ultimately cause staining failure.

9. Fixation may quench the fluorescence; do not fix the samples.

1. Wu XX, Sun Y, Zhou XB, Zhang J, Yu BY, Xu Q. Effect of Bufotalin on the Apoptosis of BGC-823 Cells. Chinese Medicine and Clinical Pharmacology of New Drugs. 2007 March, Vol.18 No.2.

2. Yang L, Zhou X, Yang J, Yin X, Han L, Zhao D. Aspirin inhibits cytotoxicity of prion peptide PrP106-126 to neuronal cells associated with microglia activation in vitro. J Neuroimmunol. 2008 Aug 13;199(1-2):10-7.

3. Gao C, Jiang Y, Tan C, Zu X, Liu H, Cao D. Synthesis and potent antileukemic activities of 10-benzyl 9(10H)-acridinones. Bioorg Med Chem. 2008 Sep 15;16(18):8670-5. Epub 2008 Aug 5.

4. Wang LM, Li QY, Zu YG, Fu YJ, Chen LY, Lv HY, Yao LP, Jiang SG. Anti-proliferative and pro-apoptotic effect of CPT13, a novel camptothecin analog, on human colon cancer HCT8 cell line. Chem Biol Interact. 2008 Nov 25;176(2-3):165-72. Epub 2008 Jul 30.

5. Wang L, Li Z, Wang C, Yang Y, Sun L, Yao W, Cai X, Wu G, Zhou F, Zha X. E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression. Arch Biochem Biophys. 2009 Jan 1;481(1):116-22. Epub 2008 Nov 1.

6. Yan HL, Xue G, Mei Q, Wang YZ, Ding FX, Liu MF, Lu MH, Tang Y, Yu HY, Sun SH. Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis. EMBO J. 2009 Sep 16;28(18):2719-32. Epub 2009 Aug 20.

7. Ji YB, Qu ZY, Zou X. Juglone-induced apoptosis in human gastric cancer SGC-7901 cells via the mitochondrial pathway. Exp Toxicol Pathol. 2009 Oct 6.

For research use only.