solution is prepared with ddH2O, filtrated with 0.22 μm filter, protected from light.

• Verapamil, 4mM(2mg/ml). add 0.002g into 1ml H2O.(re-prepare for each experiment)

• Hoechst33342(5mg/ml).(stable in a month)

• Cell dissociation. 150g, 3min

• Resuspend the medium, 150g, 3min

• Remove the supernatant, resuspend cells with 3 ml medium, and dilute to 10^6/ml

• Prepare the flow tube and mark it: ctrl, , v-100, v-200(recommended)

• Add 2 ml of suspension to each ep tube and add the corresponding volume Verapamil.

  • V-50: 25 μl

  • V-100: 50 μl

  • V-200: 100 μl

• 37 ° C water bath for 15 min (sample group does not need water bath)

• Add 2 μL of Hoechst33342 to each tube to a final concentration of 5 μg/mL

• 37 ° C water bath for 90 min, vortex every 10 min(pre-cooling the centrifuge to 4 ℃)

• Ice bath in ice box for 10min

• 4 ℃ centrifugation, 350g, 5min

• After the centrifugation, remove the supernatant

• resuspended with 500 μl cold medium, placed on ice

• Add PI (2mg/mL) 1.25μl (final concentration 5μg/ml) before loading samples, vortex, ice bath for 10min

• Filter the cell suspension into flow tubes, place on ice, protected from light.