Intracellular staining for FACS
The protocol was adopted from Krutzik and Nolan 2003 and revised
1. Start with 100,000 cells/100ul in normal medium
2. Add monensin
3. Un-adhere the cells using EDTA
4. Centrifuge and resuspend in 100μl growth medium
5. Move cells into a 96-well plate
6. Add 100μl 4% formaldehyde (Final con. Is 2% formaldehyde)
7. Fix cells for 10 min at R.T
8. Centrifuge at 1,200 rpm for 5 min
9. Wash cells with HBA (FACS buffer)
10. Discard supernatants and add 50-60ul of -20оC MeOH Permabilize cells in
MeOH for 15 min on ice
11. Wash cells with HBA
12. Centrifuge at 1,200 rpm for 5 min
13. Discard supernatants and add 100ul HBA
14. Add optimal con. of Abs
15. Incubate 30min, on ice
16. Add 100ul HBA and spin and discard supernatants.
17. Resuspend cells in 200ul of HBA in FACS tubes.
Relmα antibody con. to use: 1μg/ml
YM1 antibody con. to use: μg/ml
4о ‫אפשר לשמור ב‬