Annexin V-FITC/PI Apoptosis Kit (Suitable For Flow Cytometers Other Than C6)

Product Description

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.
Note:that this product is not suitable for automatic voltage-adjusting flow cytometers such as the BD C6; if used with such devices, we recommend using the Annexin V-FITC/PI apoptosis kit (C6-specific).

Assay Procedure

A. Adjustment of instrument parameters

1. Collect 1×10⁶ -3×10⁶ cells were centrifuged and washed twice with precooled PBS, and the supernatant was discarded.
2. Add 500 uL Apoptosis Positive Control Solution heavy suspension and incubate on ice for 30 minutes.
3. Wash centrifugally with pre-cooled PBS and discard the supernatant.
4. Add appropriate amount of precooled 1 × Binding Buffer resuspension, and add the same number of untreated living cells to mix with it. Adding precooled 1 × Binding Buffer to 1.5 mL, it was equally divided into three tubes, of which one tube was blank control tube and two tubes were single dye tube.
5. The single dye tube was incubated with 5 uL Annexin V-FITC or 10 uL PI at room temperature and away from light for 5 minutes.
6. On the flow cytometry, the voltage of FSC, SSC and fluorescence channel is reguLated by blank tube, and under this voltage condition, the voltage is reguLated by single dye tube.Compensation of fluorescence channels.

Note: some treatment of adherent cells into single cells will cause damage to the cell membrane, resuLting in false positive of Annexin V. therefore.

Optimization is needed. Enzymes that are milder to cells such as Accutase can be used

B. Sample testing

1. Apoptosis was induced according to the experimental scheme.
2. Wash centrifugally with precooled PBS and collect 1-10 × 10 5 cells (including cells in the cuLture supernatant). Dilute 5 × Binding with double distilled water.Buffer is 1 × working solution, and 500 uL 1 × Binding Buffer resuspension cells are taken.
3. 5 uL Annexin V-FITC and 10 uL PI were added to each tube.
4. After mixing gently and swirling, incubate at room temperature away from light for 5 minutes.
5. According to the experimental method, flow analysis is carried out.
6. Flow analysis.

Annexin V-FITC was detected by FITC detection channel (Ex = 488 nm; Em = 530 nm) and by PI on flow cytometry.PI was detected by Ex (535 nm; Em = 615 nm).