Protocol for Combined Staining of Intracellular Proteins and Cell Surface Markers in Blood

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. Adjust pH to 7.4. Store at room temperature.
2. Formaldehyde (methanol free).
3. Triton™ X-100
4. 50% Methanol
5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

1. Aliquot 100 µl fresh whole blood per assay tube.
2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
3. Add 65 µl of 10% formaldehyde to each tube.
4. Vortex briefly and let stand for 15 min at room temperature.
5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
6. Vortex and let stand for 30 min at room temperature.
7. Add 1 ml Incubation Buffer.
8. Pellet cells by centrifugation and aspirate supernatant.
9. Repeat steps 7 and 8.
10. Resuspend cells in 1 ml cold 50% methanol in PBS (store methanol solution at −20°C until just before use).
11. Incubate at least 10 min on ice.
12. Proceed with staining or store cells at −20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

1. Add 1 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
2. Add primary antibodies diluted as recommended on datasheet in Incubation Buffer.
3. Incubate for 30–60 min at room temperature.
4. Rinse as before in incubation buffer by centrifugation.
5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer according to the manufacturer’s recommendations.
6. Incubate for 30 min at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) 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.