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

Reagents

• Phosphate buffered saline (catalog BUF036A) containing 1%bovine serum albumin (PBS/BSA)
• Phosphate buffered saline (PBS) (BUF036A)
• Staining Buffer (BUF073)
• Required when staining whole blood:
  • Erythrolyse Red Blood Cell Lysing Buffer (10x) (BUF04), dilute to 1x using distilled water
  • Anticoagulant

Method

1. Prepare cells appropriately. For information on preparation of cells from tissue culture cell lines, tissues, and isolation of peripheral blood mononuclear cells from whole blood refer to protocol FC1, FC2, or FC3. Resuspend cells at a concentration of 1 x 10⁷ cells/ml in Staining Buffer (BUF073).[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^oC for 30-60 min, avoiding direct light.
4. Add 2 ml Staining Buffer (BUF073), centrifuge at 300-400 g for 5 min, and discard the supernatant.
5. Resuspend cells in 2 ml Staining Buffer (BUF073), centrifuge at 300-400 g for 5 min, and discard the supernatant.

Note: Another wash step can be performed to reduce background staining. [Alternatively, for whole blood perform a red cell lysis. Resuspend in 2 ml of 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF040). Incubate for 10 min at room temperature (RT). Centrifuge at 300-400 g for 5 min and discard the supernatant. Wash with 2 ml PBS/BSA, centrifuge at 300-400 g for 5 min, and discard the 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 300-400g at RT for 5 min, and discard the supernatant.
7. Resuspend cells in 200 μl cold (4^oC) PBS and store in the dark at 4oC.
8. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hours.

Notes

It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. To block human cells, we recommend using Bio-Rad’s Human Seroblock (BUF070), and for mouse cells using Bio-Rad’s Mouse Seroblock FcR (BUF041). 

Including a viability dye is recommended to allow gating on live cells during analysis. 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 Cell Viability Assay (13511), must be used.

Staining can also be performed 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 LYNX Rapid Conjugation Kits™ and Readilink Kits. Alternatively, an indirect staining approach can be used as described in protocol FC5: Indirect Immunofluorescence Staining of Surface Epitopes of Cells and Whole Blood.