Preparation of Human Peripheral Blood Mononuclear Cells

Reagents

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

Histopaque or Ficoll

Methods

1. Allow separation media such as Histopaque to equilibrate to room temperature.
2. Dilute blood in equal volumes of room temperature PBS/BSA (for example, add 3 ml of PBS/BSA to 3 ml of blood).
3. Carefully overlay whole blood onto an equal volume of separation media in a 15 ml conical centrifuge tube.
4. Centrifuge at 300-400g for 30 minutes in a 20^oC temperature controlled centrifuge with no brake. Note: Centrifugation at 4^oC or with brake reduces efficiency of cell recovery.
5. Harvest cells from the serum/separation media interface using a pipet.
6. Place harvested cells in a 15 ml conical centrifuge tube.
7. Adjust the volume to 10 ml with PBS/BSA.
8. Centrifuge at 300-400 g for 5 minutes at room temperature.
9. Discard supernatant and resuspend pellet to a final concentration of at least 1 x 10⁷ cells/ml with cold (4^oC) PBS/BSA.

Notes

• It is recommended that all containers which have come into contact with human blood or cells should be considered hazardous waste and discarded appropriately.
• 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.