PBMC flow cytometry

Protocol for obtaining PBMCs and staining for flow cytometry for CD4+ cytotoxic cells 

For obtaining PBMCs

Reagents:

• Ficoll® Paque Plus (GE Healthcare, Catalog number No. 17-1440-03)

• Dulbecco's Phosphate-Buffered Saline (Gibco, Catalog number: 21600010)

1. Collect blood in VACUTAINER tubes containing the appropriate anticoagulant (heparin, EDTA tube) in the specified volume for the experiment. Each mL of blood contains approximately 1.5 × 10⁶ PBMCs.

2. For greater efficiency in cell isolation, dilute the blood volume with an equal volume of 1X PBS. All equipment and solutions that meet the cells must be sterile.

3. Prepare the gradient for centrifugation in a 50 mL conical-bottom tube using an equal volume of Ficoll and diluted blood (9 mL of Ficoll and 9 mL of blood previously diluted in 9 mL of PBS, for a total volume of 27 mL). First, add the Ficoll, then slowly and carefully layer the blood-PBS mixture on top.

4. Centrifuge at 600g for 40 minutes at 18-20°C using a swinging-bucket rotor, without brake. Immediately after centrifugation, the centrifuge temperature should be set to 4°C.

For the isolation of PBMCs

5. Collect the PBMC layer (located at the interface between Ficoll and plasma) and transfer it to a new, sterile 15 mL conical-bottom tube.

6. Fill the tube with 1X PBS to a volume of 15 mL at 4°C to wash the cells and mix by gentle inversion.

7. Centrifuge at 400g for 10 minutes at 4°C.

8. Carefully discard the supernatant and then resuspend the pellet.

9. Repeat steps 6, 7, and 8 two more times.

10. Resuspend the cells in 1 mL of 1X PBS, count the cells, and proceed with in vitro stimulation or direct flow cytometry Staining.

11. Adjust the cell concentration to 1 × 10⁷ in 1X PBS