Protocol: Direct Immunofluorescence Staining of Intracellular Antigens Using Antibodies Labeled with StarBright™ Dyes

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Staining Buffer (BUF073)
• eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, 00-5523-00) containing:
  • Fixation/permeabilization concentrate
  • Fixation/permeabilization diluent
  • Permeabilization buffer (10x)
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• BD Horizon Brilliant Stain Buffer (BD Biosciences, 563794)
• Optional: Tween 20
• Optional: Fixable live/dead dye

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 x 10⁷ cells/mL.
3. Aliquot 100 µL of the cell suspension into as many 5 mL tubes as required.
4. Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocols FC4 and FC21 for more details.
5. Wash cells once in 2 mL cold (4°C) PBS/BSA and discard supernatant.
6. Prepare Foxp3 fixation/permeabilization working solution by mixing 1 part fixation/permeabilization concentrate with 3 parts fixation/permeabilization diluent.
7. Add 1 mL Foxp3 fixation/permeabilization working solution to each tube and vortex, ensuring that the pellet is fully dissociated. Incubate in the dark at room temperature (RT) for 30 min.
8. Prepare 1x Foxp3 permeabilization buffer by mixing 1 part permeabilization buffer (10x) with 9 parts dH₂O.
9. Add 2 ML 1x permeabilization buffer, certrifuge at 400-600 x g for 5 min at RT, and discard supernatant.
10. Repeat step 9.
11. Resuspend cells in the residual volume of permeabilization buffer, typically 100 µL, and block for 15 min at RT with the chosen blocking reagent. See the notes section for common blocking reagents.
12. Add Brilliant Stain Buffer to a total volume of 20–50%. Add the StarBright Dye–labeled antibody at the dilution determined by a titration experiment. Mix well and incubate for 60 min at RT or 18 hr (overnight) at 4°C while avoiding direct light.
13. Add 2 mL 1x permeabilization buffer to each tube, centrifuge at 400–600 x g for 5 min at RT, and discard supernatant.
14. Repeat step 13.
15. Resuspend cells in 200 µL cold (4°C) PBS and store in the dark at 4°C.
16. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• Use of a blocking reagent is strongly recommended to reduce background staining. The most effective blocking reagent is cell and antibody dependent. Typical blocking reagents are Human Seroblock (BUF070) for human cells and Mouse Seroblock FcR (BUF041) for mouse cells. Serum is also an effective blocking reagent when used at a concentration of 3–10%
• The addition of 0.1% Tween 20 during antibody incubation may also reduce nonspecific background staining
• Different incubation times have been effective for intracellular staining using StarBright Dye–conjugated antibodies, from 1 hr at RT to 18 hr (overnight) at 4°C. Use the most appropriate protocol for your experiment
• Antibodies labeled using a TrailBlazer Tag and the TrailBlazer StarBright Dye Label Kit typically stain optimally at 5 µL per test for 1 hr at RT. A lower dilution is required for overnight staining. It is best practice to titrate all antibodies in your experiment using the same conditions as in the final experiment. Further information regarding titration best practices is available on the Bio-Rad website
• To allow gating on live cells during analysis, a viability dye staining step prior to step 6 is recommended. A fixable dye such as VivaFix 353/442 Cell Viability Assay (1351111) is required for intracellular staining