Phospho-Flow Protocol: PFA/MeOH Technique
Protocol
Materials
- Cells of interest.
- Chemicals or cytokines for induction of phosphorylation.
- Formaldehyde.
- Ampules of 16% or 32% in water (Electron Microscopy Services).
- 100% methanol at 4°C.
- Antibodies – various suppliers.
- Becton Dickinson is currently developing and selling phospho-specific antibodies that are conjugated to Alexa Fluor dyes. These dyes match well to FITC and APC channels, and allow two-color experiments to be performed.
- Staining media.
- PBS (phosphate buffered saline)
- 0.5% BSA (albumin from bovine serum)
- 0.02% NaN 3 (sodium azide)
- 5 ml FACS tubes.
Experimental Protocol
- Stimulate cells. Be sure to keep an unstimulated sample as control.
- At least 1 million cells provide optimal results, though each sample can go as high as 10 million cells
- Fix cells directly with 1.5% formaldehyde for 10 min at room temperature (RT).
- Pellet cells at 500g for 5 min.
- Resuspend with vortexing in 100% methanol (about 1ml per 1 million cells).
- Incubate for 10 min to 16 hours at 4°C (or longer at –20°C).
- Pellet (as in Step 3) and wash twice with staining media.
- Resuspend cells in staining media at 10 7cells/100 ul.
- Add antibodies at optimal titration.
- Incubate for 30 min at RT.
- Wash with staining media (at least 15 volumes: ~2-3 ml for 100 ul).
- Analyze by flow cytometry.
