Protocol: Direct Immunofluorescence Staining of Cells with StarBright™ Dye-Conjugated Antibodies

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Staining Buffer (BUF073)
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock (BUF041)
• Live/dead dye
• Optional: Fixation buffer (BUF071)

Methods

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 PBS/BSA or Staining Buffer (BUF073) and adjust to 1 x 10⁷ cells/mL.
3. Aliquot 100 μL of the cell suspension into as many 5 mL tubes (or wells of a 96-well plate) as required.
4. Incubate in blocking reagent, e.g., Human Seroblock (BUF070), Mouse Seroblock (BUF041), or 10% serum of the same species as the cells you are using, for 10–30 min at room temperature (RT).
5. Centrifuge at 300–400 x g for 5 min at 4°C and discard supernatant.
6. Add antibody or multiple antibodies at the recommended dilution in 100–200 μL of staining buffer and incubate overnight at 4°C or 1 hr at RT while avoiding direct light.
   Note: As staining cells on ice will result in a lower staining index, we recommend staining at 4°C or RT to improve resolution.
7. Wash cells with 2 mL cold (4°C) Staining Buffer (200 μL for 96-well plates), centrifuge at 300–400 x g for 5 min, and discard supernatant. Repeat for a total of three washes.
8. 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.
9. Resuspend cells in 200 μL of cold (4°C) PBS and store in the dark at 4°C.
10. 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, Bio-Rad’s Mouse Seroblock FcR (BUF041) for blocking mouse cells, or 10% serum of the same species as the cells you are using
• 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
• StarBright Dye-conjugated antibodies are available as a 5 μL test but you may wish to titrate all the antibodies in your experiment for your particular cells, as it may improve the stain index
• Staining at lower temperatures generally requires longer incubation to reach antibody binding equilibrium, and this effect is more pronounced for StarBright Dyes due to their larger size. For optimum staining, Bio-Rad recommends incubating samples either overnight at 4°C or 1 hr at RT
• StarBright Dyes do not require a special buffer when combined with any other StarBright Dye, organic fluorophore, or protein-based fluorophore, including tandem dyes. However, you may need to use recommended staining buffers for certain polymer dyes when combining with one or more of these dyes
• If a directly conjugated StarBright Dye antibody is not available commercially, an alternative is to conjugate a purified format of the antibody using Bio-Rad’s TrailBlazer™ Tag and StarBright Dye Label Kit