Protocol: Direct 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, 10x (BUF04), dilute to 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 required.
• Blocking reagent: Mouse Seroblock FcR (BUF041) or Human Seroblock (BUF070)
• Fixable live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Prepare cells appropriately. Refer to Bio-Rad’s 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. Resuspend cells at a concentration of 1 × 10⁷ cells/mL in Staining Buffer (BUF073). [Prepare undiluted whole blood in an appropriate anticoagulant.]
2. Aliquot 100 μL of the cell suspension [or whole blood] into as many 5 mL tubes as required.
3. Add directly conjugated 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.
4. Add 2 mL Staining Buffer (BUF073), centrifuge at 300–400 x g for 5 min, and discard supernatant.
5. 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 in 2 mL of 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF040) and 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.]
6. Optional: Fix cells by resuspending in 200 μL Fixation Buffer (BUF071) and incubate for 20 min at RT in the dark, centrifuge at 300–400 x g at RT for 5 min, and discard supernatant.
7. Resuspend cells in 200 μL cold (4°C) PBS and store in the dark at 4°C.
8. 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, a viability dye staining step is recommended. A fixable dye such as VivaFix 353/442 Cell Viability Assay (1351111) is required for fixed cells
• When 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
• If a directly conjugated antibody is not available commercially, an alternative is to conjugate a purified format of the antibody using a conjugation kit. These are available in a wide range of formats, such as Bio-Rad’s TrailBlazer™ Tag and StarBright Dye Label Kits, LYNX Rapid Conjugation Kit™ and Readilink Kits. Alternatively, an indirect staining approach can be used as described in Bio-Rad’s protocol FC5: "Indirect Immunofluorescence Staining of Surface Epitopes of Cells and Whole Blood"