Flow Cytometry

Preparation of Human Peripheral Blood Mononuclear Cells

Preparation of Human Peripheral Blood Mononuclear Cells for Flow Cytometry

Preparation of Human Peripheral Blood Mononuclear Cells for Flow Cytometry

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This method provides a general procedure for use with peripheral blood mononuclear cells. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications.


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


  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 20oC temperature controlled centrifuge with no brake. Note: Centrifugation at 4oC 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 (4oC) PBS/BSA.


  • 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.