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. Collect blood into appropriate anticoagulant such as EDTA, heparin or acid citrate dextrose.
2. Aliquot 2-3 ml of whole blood into a 50 ml centrifuge tube.
3. Add 20-25 ml of PBS/BSA, pre-warmed to 37^oC and mix well.
4. Centrifuge at 300-400 g for 5 minutes at 37^oC. Carefully aspirate the supernatant, taking care not to disturb the cell pellet, and resuspend the pellet in the residual supernatant.
5. Repeat steps 3 and 4 twice more for a total of three washes.
6. Resuspend in 2-3 mls cold (4^oC) PBS/BSA. Aliquot 100 μl of the washed blood into the required number of test tubes. Add appropriate volume of antibody at the vendor-recommended dilution and incubate at 4^oC for at least 30 minutes, avoiding direct light.
7. Add 2 ml of freshly prepared erythrolyse red cell lysing buffer and mix well.
8. Incubate for 10 minutes at room temperature.
9. Centrifuge at 300 - 400 g for 5 minutes, discard supernatant.
10. Wash with 2 ml of room temperature PBS/BSA, centrifuge at 300-400 g for 5 minutes at room temperature and discard the supernatant.
11. Resuspend cells in 200 μl cold (4^oC) PBS or with 200 μl of 0.5% paraformaldehyde in PBS if required.
12. Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.

Notes

• The cell washing procedure described above has no effect upon other cell surface antigens, and has been shown not to affect the recovery of individual cell subsets.
• 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.