Protocol: Preparation of Peripheral Blood Mononuclear Cells for Flow Cytometry

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Separation media: Histopaque or Ficoll
• Blocking reagent: Mouse Seroblock FcR (BUF041) or Human Seroblock (BUF070)

Method

1. Allow separation media, such as Histopaque, to equilibrate to room temperature (RT).
2. Dilute blood in equal volumes of RT 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.
   Note: For volumes of blood greater than 3 mL, a 50 mL conical centrifuge tube can be used.
4. Centrifuge at 300–400 x g for 30 min in a 20°C temperature-controlled centrifuge with no brake.
   Note: Centrifugation at 4°C or with brake reduces the 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 x g for 5 min at RT and discard supernatant.
9. Resuspend cells in cold (4°C) PBS/BSA and adjust cell concentration to 1 x 10⁷ cells/mL.

Notes

• It is recommended that all containers that have come into contact with blood or cells be considered hazardous waste and discarded appropriately
• Ensure you prepare enough cells for your experiment. Typically, 10⁶ cells are used for each flow cytometry sample
• To ensure cells are in a single-cell suspension and remove debris, pass samples through a 40 μm cell strainer just prior to counting
• Count cells using a hemocytometer or an automated cell counter such as Bio-Rad’s TC20 Automated Cell Counter (1450102)
• It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. We recommend using Bio-Rad's Human Seroblock (BUF070) for blocking human cells and Bio-Rad's Mouse Seroblock FcR (BUF041) for blocking mouse cells