Protocol: Direct Immunofluorescence Staining of Intracellular Antigens: The Leucoperm Method

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Leucoperm (BUF09)
• Required when staining whole blood:
  • Erythrolyse Red Blood Cell Lysing Buffer, 10x (BUF04), dilute to 1x using distilled water
  • Anticoagulant
    Note: For basic staining, any appropriate anticoagulant, such as heparin, ethylene diamine tetraacetic acid (EDTA), or acid citrate dextrose, may be used. In some instances, specific anticoagulants may be needed.
• 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. Prepare cells appropriately and adjust cell suspension to a concentration of 1 x 10⁷ cells/mL with cold (4°C) PBS/BSA. [Prepare undiluted whole blood in an appropriate anticoagulant.]
2. Add 100 μL of cell suspension [or whole blood] into as many 5 mL tubes as required.
3. Wash cells once in 2 mL cold (4°C) 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 directly conjugated antibody at the dilution determined from a titration experiment or at 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 at 300–400 x g for 5 min, and discard supernatant.
9. Resuspend cells in 2 mL cold (4°C) PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
   Note: Another wash step can be performed to reduce background staining. [For whole blood, perform red cell lysis. Resuspend cells in 2 mL 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF04). Incubate for 10 min at RT. Centrifuge at 300–400 x g for 5 min and discard supernatant. Wash with 2 mL PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.]
10. Optional: Fix cells by resuspending in 200 μL Fixation Buffer (BUF071), incubate for 20 min at RT in the dark, centrifuge at 300–400 x g for 5 min, and discard supernatant.
11. Resuspend cells in 200 μL cold (4°C) PBS and store in the dark at 4°C.
12. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• An unconjugated primary antibody can also be used: add the primary antibody and wash (per steps 7 and 8), then repeat steps 7 and 8 using an appropriate secondary antibody. There is no requirement to add more Leucoperm (BUF071)
• 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