Reagents:

  • Ficoll-Paque Plus, 6x 500 ml, Amersham #17144003, $295*

  • Dulbecco's Phosphate Buffered Saline (DPBS), 500ml, BioWhittaker #17-512F, $5.87

  • ACK lysing buffer, 100 ml, BioWhittaker #10-548E, $10.80

  • Sterile plastic transfer pipette, 500/case, VWR #14670-114, $43

  • 50 ml conical tubes, 300/case, ISC #C-3317-6, $98

  • Optional: Tri Sodium Citrate Dihydrate (TNC), 38 g/l*

To order buffycoats from American Red Cross, Pacific Northwest Regional Blood Services in Portland: call Hospital Services at 503-284-7008 (option 1). Need Mullins Lab PO number, FedEx account number, budget number and UWATTS code.

 

Protocol:

  1. Transfer blood from bag to sterile 250 ml bottle.

  2. Dilute with DPBS to 150 ml total volume.

  3. Fill six 50 ml tubes with 12.5 ml Ficoll-Paque Plus each.

  4. Gently pipette 25 ml of the diluted cell suspension on top of each 12.5 ml of Ficoll-Paque Plus.

  5. Spin 20 minutes at 2000 rpm. No brake!

  6. Transfer the white blood cell ring fraction to a new 50 ml tube using a sterile Pasteur pipette. Combine 2 rings into one 50 ml tube.

  7. Adjust the volume to 50 ml per tube using PBS.

  8. Spin 10 minutes at 1700 rpm.

  9. Discard the supernatant.

  10. Resuspend each pellet in 2 ml ACK lysing buffer to lyse remaining erythrocytes.

  11. Incubate for no more than 2 minutes at room temperature.

  12. Adjust the volume to 50 ml using PBS.

  13. Spin 10 minutes at 1200 rpm.

  14. Resuspend the pellet in 1x PBS. At this point, cells are in two or three tubes; combine cell pellets in 10 ml PBS, rinse tubes with another 10 ml and add to the rest.

  15. Count the cells.

 

Notes:

Ficoll-Paque Plus and PBS should be at room temperature. 10% TNC (v/v) in PBS can be used if erythrocytes are a problem (e.g., with macaque blood). This protocol can also be used to get rid of dead cells: use 2.5 ml Ficoll in 15 ml conical tube, layer 10 ml cell suspension on top, follow protocol from step 5.

TNC is added to increase red blood cell aggregation and decrease monocyte adherence to the tubes. However, the US buffycoats reacted differently and the TNC was not sufficient to remove the red cells, so we included a step with LCK lysing buffer to remove them. Since monocyte yield was not a problem we decided to discontinue the use of TNC for the US buffycoats.

Department of Microbiology
School of Medicine
University of Washington
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