Direct immunofluorescence of intracellular antigens: Paraformaldehyde / Saponin method

Reagents

Phosphate buffered saline (BUF036A) containing 1% bovine serum albumin (BSA).

PBS.

4% (w/v) paraformaldehyde in PBS (Note: dissolve on heated stirrer and cool before use)

0.1% (w/v) saponin in PBS

Optional: 0.5% (w/v) paraformaldehyde in PBS (Note: dissolve on heated stirrer and cool before use)

Method

1. Prepare cells appropriately; refer to protocol FC1. ‘Preparation of cells for flow cytometry’ for further information. Adjust the cell suspension to a concentration of 1 x 10⁷ cells/ml with cold (4^oC) PBS/BSA.
2. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at 4^oC, avoiding direct light.
3. Following staining, wash cells once in 3 ml PBS/BSA pellet cells at 300-400 g and 4^oC for 5 minutes and discard supernatant.
4. Resuspend cells in 100 μl 4% paraformaldehyde per 1×10⁶ cells. Incubate for 20 minutes at room temperature. Wash once in 3 ml PBS/BSA.
5. Resuspend cells in 100 μl 0.1% saponin per 1 × 10⁶ cells. Incubate for 15 minutes at room temperature.
6. Aliquot 100 μl of cell suspension into the required number of tubes containing directly conjugated antibody at the vendor-recommended dilution. Incubate for at least 30 minutes at 4^oC, avoiding direct light.
7. Wash once in 3 ml 0.1% saponin and resuspend in 200 μl 0.5% paraformaldehyde.
8. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.

Notes

• The following should be considered when designing your Flow Cytometry experiments:
  • To avoid unspecific binding, you also need to block Fc receptors on cell types such as spleen cells, with FcR blocking reagents e.g. Bio-Rad’s Mouse Seroblock reagent (BUF041).
  • Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;
    • Isotype controls used to determine if the staining is specific
    • Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
• For all multi-color Flow Cytometry experiments it is advisable to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.