Protocol: Preparation of Peripheral Blood Mononuclear Cells for Flow Cytometry
FC2
A common sample to use in flow cytometry is peripheral whole blood. Typically, white blood cells are of interest which requires the processing of whole blood by one of two ways; either using a gradient separation to generate a peripheral blood mononuclear cell (PBMC) preparation or using the blood whole and removing the red blood cells with a red cell lysis step.
This protocol is a general procedure to generate PBMCs. A certain level of technical skill and immunological knowledge is required for the successful implementation of this technique; these are guidelines only and may need to be adjusted for particular applications.
Flow cytometry analysis may be required from cells derived from other sources. For the preparation of cells from tissue culture cells please refer to Bio-Rad’s protocol FC1. Further to this, a protocol (FC3) is also available for the preparation of cells from a variety of tissue sources including peritoneal macrophages, thymus cells, spleen cells, and bone marrow.
Reagents
- Phosphate buffered saline (PBS) (catalog BUF036A) containing 1% bovine serum albumin (PBS/BSA)
- 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 300–400g for 30 min in a 20°C temperature-controlled centrifuge with no brake.
Note: Centrifugation at 4°C 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 min at RT.
9. Discard supernatant and resuspend pellet in cold (4°C) PBS/BSA and adjust cell concentration to
1 x 107 cells/ml.
Notes
- It is recommended that all containers which have come into contact with blood or cells should 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 sample 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 to avoid nonspecific binding. To block human cells, we recommend using Bio-Rad’s Human Seroblock (BUF070) and for mouse cells we recommend using Bio-Rad’s Mouse Seroblock (BUF041).
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Flow Cytometry Basics Guide
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