Virology Core Lab Protocol
2/18/2014 DD
Isolation of Peripheral Blood Mononuclear Cells
1. Prepare six 50ml tubes with 14ml of PBS (without Ca or Mg) in each.
2. With sterile scissors cut off the tubing of one leukopak and pour 11-13 ml of blood
into each 50ml tube, making sure all six are equal in volume.
3. Gently mix the contents of one of the tubes with a 10 ml serological pipet and
carefully underlay 14 ml of Ficoll-Paque Plus (StemCell Technologies Cat #07957,) using
a clean pipet. Repeat for all tubes.
4. Centrifuge the 50ml tubes for 20 minutes at 966xg without braking @ RT.
5. Remove the PBMC band and transfer to a new sterile 50ml tube. (Partially combine
from 6 tubes  4 tubes)
6. In each tube: bring the volume up to 50ml by adding 5mM EDTA diluted in PBS
(Invitrogen Cat#AM9260G) to the cells and mix.
7. Centrifuge for 7 minutes at 400xg @ RT.
8. Decant the supernatant. Resuspend cell pellets in 5mM EDTA diluted in PBS and
combine cells from two tubes into one. (4 tubes  2 tubes)
9. In each tube: add 5mM EDTA diluted in PBS to 50ml and mix.
10. Centrifuge for 10 minutes at 180xg @ RT.
11. Decant the supernatant. As before, combine cells from the two tubes to one. Filter
with a 40 um nylon cell strainer (BD Falcon Cat # REF 352340), if necessary.
12. Add 5mM EDTA diluted in PBS to 50ml and mix.
13. Centrifuge for 10 minutes at 180xg @ RT.
14. Decant the supernatant and resuspend the pellet in 50ml of RPMI 1640 medium.
15. Count the number of viable and nonviable cells.