Indirect immunofluorescence staining of cells and blood for Flow Cytometry

Reagents

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

PBS.

Erythrolyse red blood cell lysing buffer (BUF04).

Anticoagulant (Note: for basic staining any appropriate anticoagulant, such as heparin, EDTA, or acid citrate dextrose, may be used. In some instances specific anticoagulants may be required)

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 buffer.
   [Whole blood samples may be used undiluted unless the cell count is high, e.g. as in leukemia. Use appropriate anticoagulant].
2. Aliquot 100 μl of the cell suspension [or whole blood] into as many test tubes as required.
3. Add primary antibody at the vendor-recommended dilution. Mix well and incubate at 4^oC for at least 30 minutes.
4. Wash cells with 2 ml of cold (4^oC) PBS/BSA, centrifuge at 300-400 g for 5 minutes and discard the supernatant.
   [To the blood suspension add 2 ml freshly prepared red cell lysis buffer and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 300-400 g for 5 minutes and discard the supernatant. Wash with 2 ml of PBS/BSA, centrifuge at 300-400 g for 5 minutes and discard the supernatant].
5. Add an appropriate secondary reagent at the vendor-recommended dilution. Mix well and incubate at 4^oC for at least 30 minutes, avoiding direct light.
6. Centrifuge at 300-400 g for 5 minutes at room temperature and discard the supernatant.
7. Re-suspend cells in 200 μl of cold (4^oC) PBS or with 200 μl of 0.5% paraformaldehyde in PBS if required.
8. Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.

Notes

• 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.