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 -20^oC)
• 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:

1. Add 65µl of 10% formaldehyde to 100µl of whole blood for a 4% final concentration.
2. Incubate at room temperature for 10 minutes
3. Add 1ml of 0.12% Triton X-100 solution for a final concentration of 0.1%.
4. Incubate at room temperature for 30 minutes
5. Add 1ml of cold 4°C FACS/wash buffer
6. Centrifuge cells at 500 x g for 5 minutes at room temperature, discard supernatant and resuspend pellet in 1ml of 50% ice cold methanol
7. Incubate at 4°C for 10 minutes, then keep samples on ice for further processing
8. For longer term storage keep samples at -20°C

Staining Protocol:

1. 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).
2. Resuspend pellet in 100µl of FC blocking buffer and incubate 10 minutes at room temperature
3. Centrifuge cells and remove supernatant
4. Resuspend the cell pellet in 100µl of FACS/wash buffer containing diluted primary antibody per Bethyl’s recommendation
5. Incubate 30 minutes on ice
6. Rinse the cells 2X in FACS/wash buffer by centrifugation
7. Resuspend the cell pellet in 100µl of fluorescent dye conjugated secondary antibody per Bethyl’s recommendation
8. Incubate 30 minutes on ice in the dark
9. Rinse the cells 2X in FACS/wash buffer by centrifugation. Continue to minimize exposure of cells to the light
10. Resuspend the cells in 300µl FACS/wash buffer in the dark
11. Filter cells through a strainer to remove clumps
12. 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