Before you start

Bring blood, PBS and gradient medium to room temperature (18–20 °C); cold medium gives poor separation.
Process blood within a few hours of collection (EDTA or heparin tubes).

Reagents and materials

Totals are for 10 mL of blood (+10% extra). Per-mL of blood amounts are in the middle column.

Reagent	Per mL of blood	Total
PBS, room temperature (dilution)	1 mL	1 mL
Density-gradient medium (1.077 g/mL), room temperature	1 mL	1 mL
PBS + 2% FBS (washes)	3 mL	3 mL
0.4% trypan bluefor counting	1 µL	1 µL

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Dilute the blood
Dilute blood 1:1 with room-temperature PBS and mix gently.
Layer onto the gradient
Add gradient medium to a tube (for example 15 mL in a 50 mL tube) and slowly layer the diluted blood on top (up to ~30 mL) without mixing the interface.
Centrifuge with the brake off
Spin at 400 × g for 30–40 min at room temperature with acceleration low and the brake off.
Collect the mononuclear layer
Remove most of the plasma, then collect the cloudy PBMC band at the interface with a pipette into a new tube.
First wash
Fill the tube with PBS + 2% FBS and centrifuge at 300 × g for 10 min.
Second wash (removes platelets)
Resuspend and centrifuge at 200 × g for 10 min; discard the platelet-rich supernatant.
Count
Resuspend and count with trypan blue. Typical yield is roughly 1–2 × 10⁶ PBMC per mL of healthy-donor blood.

Problem	What to check or change
No clear band	Check reagents are at room temperature and the brake was off; dilute blood more for high haematocrit.
Red cells in PBMC	Short ACK lysis (1–2 min) or repeat the 200 × g wash.
Low viability after freezing	Freeze in 90% FBS + 10% DMSO at a controlled rate; see the cryopreservation protocol.

Human blood is potentially infectious: BSL-2 practices, class II cabinet, sealed centrifuge buckets and institutional approval for human samples.