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3 December 2002, modified 9 February 2006 by Ushnal Rao
Reagents:
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Ficoll-Paque
Plus, 6x 500 ml, Amersham #17144003, $295*
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Dulbecco's
Phosphate Buffered Saline (DPBS), 500ml, BioWhittaker #17-512F, $5.87
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ACK
lysing buffer, 100 ml, BioWhittaker #10-548E, $10.80
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Sterile
plastic transfer pipette, 500/case, VWR #14670-114, $43
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50 ml
conical tubes, 300/case, ISC #C-3317-6, $98
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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:
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Transfer
blood from bag to sterile 250 ml bottle.
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Dilute
with DPBS to 150 ml total volume.
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Fill
six 50 ml tubes with 12.5 ml Ficoll-Paque Plus each.
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Gently
pipette 25 ml of the diluted cell suspension on top of each 12.5 ml of Ficoll-Paque Plus.
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Spin
20 minutes at 2000 rpm. No brake!
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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.
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Adjust
the volume to 50 ml per tube using PBS.
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Spin
10 minutes at 1700 rpm.
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Discard
the supernatant.
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Resuspend
each pellet in 2 ml ACK lysing buffer to lyse remaining erythrocytes.
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Incubate
for no more than 2 minutes at room temperature.
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Adjust
the volume to 50 ml using PBS.
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Spin
10 minutes at 1200 rpm.
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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.
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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.
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