Isolation of mononuclear cells (MNC) from Buffy coats
1. Dilute Buffy coats 1:4 with PBS (usually 10 ml blood + 30 ml PBS). Buffy coats
were purchased from the blood bank, Tel Hashomer hospital (Tel. No: 03-5300436)
2. Add 10 ml of Ficoll-Hypaque (GE-Healthcare cat. No. 17-1440-03, was ordered
from the general storehouse) to the bottom of the tube insert Pasteur pipette to the
bottom of the tube and inject the Ficoll-Hypaque into the pipette by a 10 ml syringe
and 0.8x40 mm needle.
3. Centrifuge the tubes at 700g (1600 rpm) in a sorvall centrifuge, departmental
equipment, for 30 min. at RT with brakes off.
After the centrifugation 4 layers should be seen, separate from each other: about 25
ml of sup, thin white layer of the MNC, few ml of Ficoll (transparent), and in the
bottom - dark red layer of the erythrocytes and granulocytes. See figure 1
4. Remove the cellular white fraction from all tubes to one 50 ml tube: remove most
of the sup and collect gently and slowly the thin white layer with a 5ml pipette
5. Wash 3 times with 50 ml cold PBS: once at 350g (1200 rpm) for 15 min. and twice
at 230g (900 rpm) for 7-10 min. It's recommended to resuspend first with 10 ml PBS
and then complete to 50 ml.
6. Remove sup and resuspend with RPMI 1640 containing 10% serum.
7. Place the tube for 16h at 4oC to allow the separation of the MNC (pellet) from the
thrombocytes (fluid).
8. Discard the fluid gently, add 10 ml RPMI with 2% FBS, resuspend and count with
1:10 Trypan Blue (take 10µl from the tube and 90µl TB to a 96-well, resuspend and
take 10 µl from the mixture for counting).You can dilute the suspension 1:10 before
counting with TB if the conc. is too high. Usually the yield AT this point is ~
5x107/ml MNC (total of 5x108).