Intracellular Staining Protocol
1. Fix cells- Add16% formaldehyde directly into culture medium to obtain a final concentration of 1.5% formaldehyde.
2. Incubate in fixative for 10 min at RT and pellet.
3. Permeabilize cells- Resuspend pellet with vigorous vortexing in 500 ul ice-cold MeOH per 10^6 cells. This is approximate number, more or less MeOH cna be used as long as evaporation is not significant.
4. Incubate at 4C for at least 10 min. At this point cells can be stored at -20C for several weeks with minor loss in signal strength.
5. Wash cells twice in staining media (PBS containing 1% BSA) then resuspend in staining media at .5-1x106 cells per 100ul.
6. Stain cells- Add optimal concentrations of antibody and incubate for 15-30 min at RT.
7. Wash cells in 15 volumesof staining media and pellet.
8. Resuspend samples in stainging media and analyze.
-This protocol was taken from Peter Krutzik and Garry Nolan in Intracellular Phopho-protein Staining Techniques for Flow Cytometry: Monitoring Single Cell Signaling Events. Cytometry 55A:61-70
-A titration of the staining antibody is important to do because this will decrease background staining and increase signal to noise ratio.
-Slight variations from this protocols may be necessary with your cell types. Things like fixing time, temp, and concentration might need to be altered. However the above protocols showed good results with pERK,pp38, pJNK, pStat1, pStat5 and p Stat6 and is probably suficient for most intracellular anitgens.
-Choose optimal fluorochrome. Because of its size, avoid PE. Most autofluoresence of cells occurs in FITC channel and the autofluoresence increases when cells are fixed. Therefore try to avoid FITC or Alexa 488.