Protocols
Fixation and Permeabilization of Whole Blood with Red Blood cell Lysis
Reagents Required:
- 10% Formaldehyde (Methanol Free)
- 0.12% Triton X-100 in PBS
- 50% Methanol in PBS (stored at -20oC)
- FACS/wash buffer: 4% Fetal Bovine Serum (FBS) or 2% Bovine Serum Albumin in PBS
- FC blocking buffer: FC block diluted per manufacturer instructions (BD Biosciences 564219)
Cell Fixation and Permeabilization Protocol:
- Add 65µl of 10% formaldehyde to 100µl of whole blood for a 4% final concentration.
- Incubate at room temperature for 10 minutes
- Add 1ml of 0.12% Triton X-100 solution for a final concentration of 0.1%.
- Incubate at room temperature for 30 minutes
- Add 1ml of cold 4°C FACS/wash buffer
- Centrifuge cells at 500 x g for 5 minutes at room temperature, discard supernatant and resuspend pellet in 1ml of 50% ice cold methanol
- Incubate at 4°C for 10 minutes, then keep samples on ice for further processing
- For longer term storage keep samples at -20°C
Staining Protocol:
- Centrifuge 1.0 x 106 cells at 500 x g for 5 minutes at 4°C and discard supernatant (perform all subsequent spins at these conditions).
- Resuspend pellet in 100µl of FC blocking buffer and incubate 10 minutes at room temperature
- Centrifuge cells and remove supernatant
- Resuspend the cell pellet in 100µl of FACS/wash buffer containing diluted primary antibody per Bethyl’s recommendation
- Incubate 30 minutes on ice
- Rinse the cells 2X in FACS/wash buffer by centrifugation
- Resuspend the cell pellet in 100µl of fluorescent dye conjugated secondary antibody per Bethyl’s recommendation
- Incubate 30 minutes on ice in the dark
- Rinse the cells 2X in FACS/wash buffer by centrifugation. Continue to minimize exposure of cells to the light
- Resuspend the cells in 300µl FACS/wash buffer in the dark
- Filter cells through a strainer to remove clumps
- Analyze with a flow cytometer
Portions of this protocol adapted from: Chow S. et al. . Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67, 4–17 (2005). PMID16080188