[민유홍] ApopTag staining protocol for FACS analysis (CHEMICON)

ApopTag staining protocol for FACS analysis (CHEMICON)

 

Assay Set-Up

4% BSA in PBS

0.5 ml and 0.2 ml PCR tubes coated with 4% BSA in PBS (add sufficient 4% BSA in PBS into tubes then rotate tubes ON at 4℃),

PCR machine

RNase A (10 mg/ml)

 

For HCT116 cells cultured in 6 well plate

1. harvest cells including media with 15ml conical tubes

   400 X g, 5 min, swinging rotor

2. resuspend pellet completely by pipetting in 300 ul of PBS, then add 100 ul of 4% paraformaldehyde in PBS and mix

3. fix for 15min on ice or ON at 4℃

4. spin down (400 X g, 5 min, swinging rotor) and resuspend in 0.5ml of PBS

5. transfer cells into 0.5 ml PCR tubes coated with 4% BSA in PBS

6. spin down (6000 ~ 7000 rpm, 5 min, HANIL Micro-12)

7. resuspend in 50 ul of 4% BSA in PBS completely, then add 150ul of permeabilization buffer (0.1% sodium citrate, 0.1% Triton X-100) and mix

8. incubate for more than 30 min (about 45 min) at 70 ~ 72 ℃.

   During incubation, resuspend cells by vortexing as often as possible

9. cool the tubes at RT, then add 200 ul of 4% BSA in PBS and ~ 4 ul of RNase A (10 mg/ml)

10. rotate ON at 4℃

11. spin down (6000 ~ 7000 rpm, 5 min, HANIL Micro-12)

12. resuspend in 200 ul of PBS, then transfer into 0.2 ml PCR tubes coated with 4% BSA in PBS

13. spin down (6000 ~ 7000 rpm, 5 min, HANIL Micro-12)

14. resuspend the cells in 40 ul EQUILIBRATION BUFFER

15. spin down, then remove supernatant

16. resuspend in 25 ul of WORKING STRENGTH TDT ENZYME

17. incubate for 1 hour at 37 ℃

 During incubation, resuspend cells by pipetting often (about 3 times)

18. spin down, then remove supernatant

19. resuspend in 200 ul of WORKING STRENGTH STOP/WASH BUFFER

20. spin down, then remove supernatant

21. wash with 200 ul of PBS once

22. resuspend 50 ul of anti-c-myc antibody (9E10) solution 1:100 diluted in 0.5 % BSA, 0.05% Triton X-100 in PBS

23. incubate for 1.5 hour at RT

 During incubation, resuspend cells by pipetting often (about 3 times)

24. wash twice with 200 ul of 0.05% Triton X-100 in PBS

25. resuspend in 50 ul of WORKING STRENGTH ANTI-DIGOXIGENIN-FLUORESCEIN containing anti-mouse ALEXA 555

26. incubate for 1 hour at RT. Avoid exposure to light.

During incubation, resuspend cells by pipetting often (about 3 times)

27. wash once with 200 ul of 0.05% Triton X-100 in PBS

28. resuspend the cells in 500 ul of 0.05% Triton X-100 in PBS

29. analyze the samples