Apoptosis kit V13241 and V13245 detection steps

1. Induction of apoptosis by an effective method; a negative control without induction.

2. Cells were harvested after incubation and washed with cold PBS.

3. Preparation of 1 x annexin V binding buffer: Add 1 mL of reagent C to 4 ml of deionized water, based on the amount of 10 analyses.

4. Preparation of 100 μg/mL PI working solution: Dilute 5 μl of Reagent B with 45 μl of 1×annexin V binding buffer, and the remainder can be stored for later use.

5. Centrifuge the cells collected in step 2 again, discard the supernatant, and resuspend in 1×annexinV binding buffer. Count and dilute to approximately 1 × 106 cells/mL with the above buffer. Prepare well by using 100 μl per experiment.

6. Add 5 μl of reagent A and 1 μl of the PI working solution prepared in step 4 to each 100 μl of the cell suspension.

7. Incubate for 15 min at room temperature.

8. At the end of the incubation, add 400 μl of 1×annexin V binding buffer, mix gently and place on ice.

9. Analyze stained cells as soon as possible. The 530 nm excitation light can be detected by FL1, and the excitation light of >575 nm is detected by FL3. The cell population will be divided into three groups: live cells show weak fluorescence, apoptotic cells show green fluorescence, and dead cells show red and green fluorescence at the same time.

10. The results can be verified by fluorescence microscopy and used for the detection of FITC, TRITC or Texas Red® filters.