Apoptosis assay

1.   A431 cells were seeded in a 6-well plate at 2.0 × 10 ⁵ cells per well for 48 h.

2.   The medium was replaced with 1.9 mL of fresh DMEM culture medium containing materials with designed concentrations.

3.   After incubation for 24 h, the cells were washed twice with 2 mL fresh medium, and incubated in 2 mL medium.

4.   The plate was irradiated using a 660-nm LED light at a power density of 20 mW cm⁻² for 30 s.

5.   After an additional 1 h of incubation in the dark, collect the detached cells in the medium, and transfer to a 5 mL-centrifuge tube.

6.   Wash the remaining cells in the 6-well plate twice with 2 mL PBS.

7.   800 µL of trypsin-EDTA (0.25%) was added to each well to digest the cells at 37ºC for 3 min until the cells were fully detached from the plate.

8.   Gently suspend the cells in the trypsin solution, transfer the cell suspension to the centrifuge tube with the previous cells mentioned in step 5.

9.   Centrifuge to separate the cells at 1500 rpm for 5 min and suspend the cells in PBS at a concentration of 1 × 10 ⁶ cells/mL.

10.  Transfer 100 µL of the PBS in step 9 (containing 1 × 10 ⁵ cells) to a 2 mL-centrifuge tube. Centrifuge at 1500 rpm for 5 min to precipitate the cells.

11.  The cells were suspended in 195 µL of binding buffer from the analysis kit.

12.  Add 5 µL of Annexin V–FITC and 10 µL of PI from the analysis kit.

13.  The cells were incubated under dark conditions at room temperature for 15 min and placed in an ice bath.

14.  The cells were analyzed with a MACSQuant flow cytometer. The measurement need to be complete within 1 h. The data were analyzed and processed with FlowJo 7.6.

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