PBMC Preparation from Buffy Coats

Last Updated 11/30/00

Contacts: Steve De Rosa (derosa@fhcrc.org) Ometa Herman (ometa@stanford.edu)

Materials

Qty

Location

Order Info

Culture Media

50mL

B-008 Cold room

 

hRPMI

400mL

B-008 Cold room

See Herzenberg Lab Media Preparation for details

Ficoll-Paque

60mL

B-013 Fridge 6

Amersham 17-0840-03

Penicillin G

 

B-010 Freezer 4

Sigma P-3032

Streptomycin Sulfate

 

B-010 Freezer 4

Sigma S-9137

L-Glutamine

 

B-010 Freezer 4

GibcoBRL 21051-024

50mL tubes

12

 

 

Sterile transfer pipets

2

 

 

Fenwal Plasma Transfer Set

1

B-012 Under TC hood

Baxter 4C2240 with spike and needle adapter

Buffy coat

 

Stanford Blood Center

 

 

Note: all steps done at RT

 

  1. Drain entire buffy into one 50mL tube (each buffy will usually contain ~24mL) using Fenwal Plasma Transfer Set.
  2. Split the buffy evenly into 4 50mL tubes (~6mL each)
  3. Add hRPMI (w/o FCS) media to a total volume of 35mL in each tube. We use serum-free media at this point because we have noticed occasional gel formation when serum is in the media for the Ficoll step. After the ficoll step, media with or without FCS may be used.
  4. Prepare 4 tubes of Ficoll, placing 15mL in each tube.
  5. Very slowly overlay the entire 35mL of buffy over the Ficoll.
  6. Spin RT, 2000rpm, for 20 minutes
  7. Remove the PBMC layer using a sterile transfer pipet. Distribute evenly into 2 50mL tubes.
  8. Fill each tube to 50mL with hRPMI media (w/o FCS)
  9. Spin RT, 1000rpm, for 10 minutes
  10. Aspirate, leaving pellet and resuspend in 50mL hRPMI media (w/o FCS)
  11. Spin RT, 1500rpm, for 10 minutes
  12. Aspirate, leaving pellet. Pool cells into one 50mL tube, resuspending in 30mL hRPMI media (w/o FCS)
  13. Spin RT, 1500rpm, for 10 minutes
  14. Resuspend cells in 50mL culture media.
  15. Count cells using hemocytometer.
  16. Store PBMC in tissue culture until use.

Recipes:

Culture media: RPMI 1640 w/o FCS, w/o glutamine

- 10% FCS
            - 2mL stock penicillin/streptomycin (red top 15mL tube in common equipment freezer)
- 2mL stock glutamine (blue top 15mL tube in common equipment freezer)

 

Penicillin/Streptomycin stock

            1.3g Penicillin G

            1.4g Streptomycin Sulfate

            200mL distilled water

            Stir in graduated cylinder, filter, freeze in aliquots.