1. Collect 3-4 tubes of blood in heparin vacutainers with green top (check expiration date!). You should get 1-2 million PBMC per ml of blood.

2. Use lymphoprep to isolate PBMCs.

• Dilute whole blood 1:1 with sterile 0.9% saline.
• Add diluted blood to the top of lymphoprep carefully (6ml diluted blood on top of 3 ml lymphoprep in 15ml tubes).
•  Cap and parafilm the tubes, and spin at 800xg 20 min, 20°C, NO BRAKES.
  • If blood was stored at RT more than 2 hours, increase to 30 min spin.
• Remove lymphocyte layer with a Pasteur pipette (concentric circles with pasteur pipette in pipette-man).
•  Dilute 1:1 in 0.9% saline to wash; spin 250xg 10min to pellet cells.
•  Resuspend in 0.9% saline, count cells, wash again.

3. After a few washes stain PBMC in 100 to 200 ul of facs buffer (1%BSA, PBS, 0.5mM EDTA).

• Check quickly on our lsrii if the stain is good before you facs sort.
•  You can pre-block with serum but not necessary as non-specific staining is very little.

3. Sorting on Aria- keep the cells concentrated at 20 million cells/ml and sort at low flow rate but go up to 20,000 events on the 70um nozzle. If you dilute the sample too much then you have to go up on the flow rate, which is not a good thing.

• This would allow you to sort nearly a million cells in 1 minute. So if you start with 20-30 million PBMC you should be done in 30 minutes and get about 1 million naive cells for 10 million PBMC.
• Sort the cells into 50%FCS. Then good to go.