Protocol: Direct Immunofluorescence Staining of Intracellular Cytokines in Whole Blood

Reagents

• Blood sample containing heparin as the anticoagulant
• Required when stimulating whole blood:
  • Cell Stimulation Reagent (with Brefeldin A) (500x) (BUF077)
  • Cell culture medium, additive free
• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Leucoperm (BUF09)
• Erythrolyse Red Blood Cell Lysing Buffer (10x) (BUF04), dilute to 1x using distilled water
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073)
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• Fixable live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Stimulate whole blood collected in heparin as required. It is recommended to dilute whole blood 1:1 in cell culture media and treat with reagents; include unstimulated controls.
   
   To stimulate cells, dilute 500 µL whole blood with 500 µL cell culture media. Add 2 µL Cell Stimulation Reagent (with Brefeldin A) (BUF077) to each mL of cell suspension. Incubate for 4–6 hr at 37ᵒC in a 5% CO₂ atmosphere.
    
2. Add 200 µL of the prepared cells into as many 5 mL tubes as required.
3. Wash cells once in 2 mL cold (4ᵒC) PBS/BSA and discard supernatant.
4. Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocol FC4 for more details.
5. Resuspend cells in 100 μL room temperature (RT) Leucoperm (BUF09) Reagent A (fixation medium). Incubate for 15 min at RT.
6. Add 3 mL PBS/BSA, centrifuge for 5 min at 300–400 x g, and discard supernatant.
7. Add 100 μL Leucoperm (BUF09) Reagent B (permeabilization medium). Immediately add the directly conjugated antibody at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at RT for 30–60 min while avoiding direct light.
8. Add 3 mL PBS/BSA, centrifuge for 5 min at 300–400 x g, and discard supernatant.
9. Resuspend in 2 mL of 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF04). Incubate for 10 min at RT.
10. Centrifuge at 300–400 x g for 5 min and discard supernatant.
11. Wash with 2 mL PBS/BSA, centrifuge for 5 min at 300–400 x g, and discard supernatant.
12. Optional: Fix cells by resuspending in 200 µL Fixation Buffer (BUF071) and incubate for 20 min at RT in the dark, centrifuge for 5 min at 300–400 x g, and discard supernatant.
13. Resuspend cells in 200 μL cold (4ᵒC) PBS and store in the dark at 4ᵒC.
14. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• Blood samples must be collected into heparin anticoagulant tubes. Ethylenediaminetetraacetic acid (EDTA) interferes with the cell stimulation process and therefore must be avoided
• Other cell stimulation reagents and protein transport inhibitors may be more suitable for your assay. Cell Stimulation Reagent (BUF077) contains phorbol 12-myristate 13-acetate (PMA) and ionomycin to stimulate cells and Brefeldin A as the protein transport inhibitor. Other common stimulation reagents include lipopolysaccharide (LPS), phytohemagglutinin (PHA), concanavalin A (ConA), and CD3/CD28 antibodies or beads. Common protein transport inhibitors that can be used with these alternatives are Monensin (1,000x) (BUF074) and Brefeldin A (1,000x) (BUF075)
• Culturing cells in the presence of Brefeldin A for more than 12 hr should be avoided because cell viability may be affected
• An unconjugated primary antibody can also be used. Add the primary antibody and wash (as per steps 7 and 8), then repeat steps 7 and 8 using an appropriate secondary antibody. There is no requirement to add additional Leucoperm (BUF09)
• It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. We recommend using Bio-Rad's Human Seroblock (BUF070) for blocking human cells and Bio-Rad's Mouse Seroblock FcR (BUF041) for blocking mouse cells
• To allow gating on live cells during analysis, a viability dye staining step prior to step 5 is recommended. A fixable dye such as VivaFix 353/442 Cell Viability Assay (1351111) is required for intracellular staining
• All antibodies should be titrated prior to use to ensure the optimal concentration is used