Protocol: Indirect Immunofluorescence Staining of Surface Epitopes of Cells and Whole Blood

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Staining Buffer (BUF073)
• Required when staining whole blood:
  • Erythrolyse Red Blood Cell Lysing Buffer (10×) (BUF04), dilute 1x using distilled water
  • Anticoagulant
    Note: For basic staining, any appropriate anticoagulant, such as heparin, EDTA, or acid citrate dextrose, may be used. In some instances, specific anticoagulants may be needed.
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• Live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Prepare cells appropriately. Refer to protocol FC1 for information on preparation of cells from tissue culture cell lines, FC2 for isolation of peripheral blood mononuclear cells from whole blood, and FC3 for preparation of cells from tissues.
2. Resuspend cells in Staining Buffer (BUF073) and adjust to 1 × 10⁷ cells/mL. [Use undiluted whole blood in an appropriate anticoagulant.]
3. Aliquot 100 μL of cell suspension [or whole blood] into as many 5 mL tubes as required.
4. Add unconjugated or biotin-labeled primary antibody at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4°C for 30–60 min.
5. Add 2 mL Staining Buffer (BUF073), centrifuge at 300–400 x g for 5 min, and discard supernatant.
6. Resuspend cells in 2 mL Staining Buffer (BUF073), centrifuge at 300–400 x g for 5 min, and discard supernatant. [For whole blood, perform red cell lysis. Resuspend in 2 mL of 1 x Erythrolyse Red Blood Cell Lysing Buffer (BUF040). Incubate for 10 min at room temperature (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.]
7. Resuspend cells in 100 μL Staining Buffer (BUF073). Add an appropriate conjugated secondary antibody at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4°C for 30–60 min while avoiding direct light.
8. Centrifuge at 300–400 x g for 5 min at RT and discard supernatant.
9. Resuspend cells in 200 μL Staining Buffer (BUF073), centrifuge at 300–400 x g for 5 min, and discard supernatant.
10. Optional: Fix cells by resuspending in 200 μL Fixation Buffer (BUF071) 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

• 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, including a viability dye is recommended. The most appropriate live/dead dye is dependent on the fluorophores used and if the cells are to be fixed. For fixed cells, a fixable dye, such as VivaFix 353/442 Cell Viability Assay (1351111), must be used
• For staining in 96-well plates, adjust wash volumes to 200 μL
• All antibodies should be titrated prior to use to ensure the optimal concentration is used
• Visit bio-rad-antibodies.com/secondaries-in-flow for advice on selecting the appropriate secondary detection reagent