Annexin V-FITC/PI Apoptosis Detection Kit

20T, 50T, 100T

Flow cytometry; fluorescence microscopy

Annexin V-FITC / propidium iodide double staining

FITC maximum excitation/emission: 488/525 nm. PI-DNA maximum excitation/emission: 535/615 nm.

Shipped with ice packs. If used repeatedly within a short period, store at 4 °C protected from light.

1 year

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.

FITC-labeled Annexin V can be used as a probe to detect the occurrence of apoptosis by fluorescence microscopy or flow cytometry. Propidium iodide (PI) 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, PI can penetrate the cell membrane and stain the nucleus red. Double staining with Annexin V and PI therefore allows cells at different stages of apoptosis to be distinguished.

1. Trypsin (EDTA-free)

2. PBS

3. Micropipettes

4. Low-speed centrifuge

5. Flow cytometer

1. Sample staining

(1) Suspension cells: collect the cells by centrifugation at 300 × g for 5 min at 4 °C.

Adherent cells: digest with EDTA-free trypsin and then collect the cells by centrifugation at 300 × g for 5 min at 4 °C. Avoid prolonged trypsin digestion, which may cause false-positive results.

(2) Wash the cells twice with pre-chilled PBS, centrifuging at 300 × g for 5 min at 4 °C each time, and collect 1 × 10⁵–5 × 10⁵ cells.

(3) Aspirate the PBS and resuspend the cells in 100 µl of 1× Binding Buffer (diluted with double-distilled water).

(4) Add 5 µl of Annexin V-FITC and 5 µl of PI Staining Solution and mix gently.

(5) Incubate for 10–15 min at room temperature protected from light.

(6) Add 400 µl of 1× Binding Buffer, mix well, keep on ice, and analyze the sample by flow cytometry or fluorescence microscopy within 1 hour.

2. Sample analysis

A. Flow cytometric analysis:

FITC has a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 525 nm; the PI–DNA complex has a maximum excitation wavelength of 535 nm and a maximum emission wavelength of 615 nm. Analyze the data with software such as CellQuest and plot a bivariate scatter diagram with FITC on the x-axis and PI on the y-axis. In a typical experiment, the cells can be divided into three subpopulations: viable cells show only very weak autofluorescence, early apoptotic cells show only strong green fluorescence, and late apoptotic cells are double-stained with both green and red fluorescence.

B. Fluorescence microscopic analysis:

(1) Suspension cells: place a drop of the Annexin V-FITC/PI double-stained cell suspension on a microscope slide and cover the cells with a coverslip.

Adherent cells: the cells may be cultured directly on a coverslip and apoptosis then induced.

(2) Observe the cells under a fluorescence microscope with a dual-color filter set. Early apoptotic cells show only green fluorescence, whereas late apoptotic cells are double-stained with both green and red fluorescence.

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. Propidium iodide (PI) is toxic. Wear gloves when handling it and avoid contact with skin, eyes, and mucous membranes.

4. Annexin V-FITC 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 × 10⁵.

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

7. For adherent cells, trypsin digestion damages the cell membrane and leads to a higher rate of false positives, while scraping with a cell scraper causes the cells to clump together and interferes with detection.

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

Shipped with ice packs. If the kit is to be used repeatedly within a short period of time, it may be stored at 4 °C protected from light. Valid for one year.

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For research use only.