This protocol provides a basic guide for the isolation of peripheral blood mononuclear cells (PBMC) from whole blood and the isolation of splenocytes from spleen. The recovered preparations contain myeloid cells, lymphocytes, erythrocytes, and platelets.
To Isolate PBMC from Whole Blood
Reagents Required
- Anti-coagulant (e.g., heparin, EDTA, or sodium citrate)
- Sterile cell culture medium, phosphate buffered saline (PBS), or other physiological balanced salt solution
- Ficoll-Hypaque™ or other suitable density medium.
Materials Required
- Sterile materials for collecting blood (e.g., syringe, tubing, needle)
- Sterile 50 mL or 15 mL centrifuge tubes
- Sterile pipettes
- Hemocytometer
- Benchtop centrifuge
- Microscope
Procedure
- Perform these steps using sterile solutions and aseptic technique.
- Collect blood sample with anti-coagulant. Or use commercially obtained, anti-coagulant treated blood (e.g., Leukopak).
- Mix blood with an equal volume of sterile PBS or other balanced salt solution. Wash cells by centrifuging at 400 x g for 10 minutes at room temperature. Carefully aspirate and discard the upper layer of plasma. Resuspend the cells in 30 mL (total volume) of sterile PBS.
- Add 20 mL of room temperature (21 °C to 23 °C) Ficoll to a 50 mL centrifuge tube. Carefully layer 30 mL of the cell suspension on top of the Ficoll. Avoid mixing the cell suspension and Ficoll layers.
- Centrifuge at 800-850 x g for 30-40 minutes at room temperature with no braking. The sample will be separated into layers as indicated in Figure 1.
- Carefully aspirate and discard the upper layer.
- Recover buffy coat layer at the top of the Ficoll layer. This layer contains peripheral blood mononuclear cells (PBMC) which are predominantly lymphocytes and monocytes.
- Wash PBMC one time with PBS to remove any residual separation media. This can be accomplished by spinning the cells for 10 minutes at 400 x g.
- To remove red blood cells, process the cell pellet with Human Erythrocyte Lysing Kit (Catalog # WL1000) according to the package instructions. Briefly, disrupt the cell pellet by “racking” the tube, resuspend the cells in erythrocyte lysis solution, vortex the cell suspension, incubate the cell suspension for 10 minutes at room temperature, and wash the cells with Wash Buffer from the lysing kit.
- Wash the cell pellet with PBS by centrifuging at 200 x g for 5 minutes.
- Suspend PBMC in buffer or medium and count cells for analysis, cell culture, or further enrichment of cell populations.
To Isolate Splenocytes from Spleen
Reagents Required
- Sterile cell culture medium, phosphate buffered saline (PBS), or other physiological balanced salt solution
- Benchtop centrifuge
- Microscope
Materials Required
- Sterile surgical scissors and forceps
- Sterile stainless steel mesh
- Sterile 100 mm Petri dish
- Sterile pipettes
- Hemocytometer
Procedure
- Perform these steps using sterile solutions and aseptic technique.
- Remove spleen from a mouse, rat, or rabbit.
- Place spleen in plastic dish with sterile PBS or cell culture medium.
- Tease apart spleen with forceps and pass through stainless steel mesh to obtain a single cell suspension.
- Wash splenocytes once with sterile PBS.
- To remove red blood cells, process the cell pellet with Mouse Erythrocyte Lysing Kit (Catalog # WL2000) according to the package instructions. Briefly, disrupt the cell pellet by “racking” the tube, resuspend the cells in erythrocyte lysis solution, vortex the cell suspension, incubate the cell suspension for 5-10 minutes at room temperature, and wash the cells with Wash Buffer from the lysing kit.
- Suspend splenocytes in buffer or medium and count cells for analysis, cell culture, or further enrichment of cell populations.
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CD4+ T Cell Isolation from PBMC or Splenocyte Preparations by T Cell Enrichment Column
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Mouse and Human Dendritic Cell Subsets