Protocol: Direct Immunofluorescence Staining of Intracellular Antigens: The Digitonin Method

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• PBS containing 0.05% (v/v) Tween 20 (PBS/Tween)
• 10 µg/mL digitonin in PBS
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073)
• 0.5% (w/v) paraformaldehyde in PBS
  Note: Dissolve on heated stirrer and cool before use.
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• Fixable Live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Prepare cells appropriately and adjust cell suspension to a concentration of 1 x 10⁷ cells/mL with cold (4ᵒC) PBS/BSA.
2. Add 100 μL of the cell suspension into as many 5 mL tubes as required.
3. Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocol FC4 for more details.
4. Add 2 mL cold (4ᵒC) PBS/BSA, centrifuge for 5 min at 300–400 x g and 4ᵒC, and discard supernatant.
5. Repeat wash step 4.
6. Add 50 μL PBS/BSA to each tube, followed by 100 μL 0.5% paraformaldehyde and incubate for 20 min at room temperature (RT).
7. Add 3 mL PBS/Tween 20, centrifuge for 5 min at 300–400 x g, and discard supernatant.
8. Repeat wash step 7.
9. Add 100 μL cold (4ᵒC) digitonin. Add the directly conjugated antibody at the dilution determined from a titration experiment or the vendor-recommended dilution and incubate for at least 30 min at 4ᵒC while avoiding direct light.
10. Add 3 mL PBS/Tween 20, centrifuge for 5 min at 300–400 x g and 4ᵒC, and discard supernatant.
11. Repeat wash step 10 twice.
12. Optional: Fix cells by resuspending in 200 µL Fixation Buffer (BUF071) and incubate for 20 min at RT in the dark, centrifuge for 5 min at 300–400 x g, and discard supernatant.
13. Resuspend cells in 200 μL cold (4ᵒC) PBS and store in the dark at 4ᵒC.
14. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• This procedure causes a reduction in the forward scatter (FSC) signal from cells, so the FSC settings on the flow cytometer may need to be adjusted
• 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 prior to step 5 is recommended. A fixable dye such as VivaFix 353/442 Cell Viability Assay (1351111) is required for intracellular staining
• All antibodies should be titrated prior to use to ensure the optimal concentration is used