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

IMPORTANT: Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in Flow Cytometry (F). This protocol is provided as a general approach for combined staining in whole blood, but CST does not validate all Flow approved antibodies for use with this protocol. Please see the product-specific Flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS) (#9808): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
2. 16% Formaldehyde (methanol free).
3. Triton X-100: To prepare 50 ml of 0.1% Triton X-100 add 50 μl Triton X-100 to 50 ml 1X PBS and mix well.
4. 50% methanol.
5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
6. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat (#4416, #4418).

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 ice-cold 50% methanol in PBS (store methanol solution at -20°C until 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

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

1. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
3. Incubate for 30–60 min at room temperature.
4. Wash by centrifugation in 2–3 ml incubation buffer. If using a directly conjugated antibody, skip to step 8.
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. Wash by centrifugation in 2–3 ml incubation buffer.
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.