Alternate Protocol for Signaling Analysis in Peripheral Blood Labeled with Surface Markers

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently 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. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 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 minutes at room temperature.
5. Add 1 ml of Triton X-100 in PBS to make 0.1% Triton final concentration.
6. Vortex and let stand for 30 minutes at room temperature.
7. Add 1 ml Incubation Buffer.
8. Pellet cells by centrifugation and aspirate supernatant.
9. Resuspend cells in cold 50% methanol in PBS (store methanol solution at –20°C until just before use).
10. Incubate at least 10 minutes on ice.
11. 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 minutes 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 minutes 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(1), 4–17.

*Recommended Secondary Antibodies:

Anti-Rabbit

• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414

Anti-Mouse

• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4409
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410

Anti-Rat

• Anti-Rat IgG (H+L), (Alexa Fluor^® 488 Conjugate) #4416
• Anti-Rat IgG (H+L), (Alexa Fluor^® 555 Conjugate) #4417
• Anti-Rat IgG (H+L), (Alexa Fluor^® 647 Conjugate) #4418