Dilute the fresh blood 1:1 with PBS* at room temperature (RT).
Caution: blood hazard !! Hepatitis B/ AIDS hazard!! Therefore be extremely careful
with human blood !! Get Hepatitis B vacination !! Try to wear double latex gloves.
All soluble wastes have to be decontaminated. Use bleach or a different
decontaminant.
Dispense 20 ml of Ficoll-Paque** in sterile 50 ml falcon tubes and overlay 1:1 with diluted blood (important: phases should not mix !!) to separate the lymphocytes and monocytes from the erythrocytes and blood-plasma.
Centrifuge in Sorvall table top centrifuge model: RT 6000B at RT at 1800 rpm for 25 min brake off (, because of ficoll- gradient !!).
5. Carefully take interphase out with a pipette and transfer it to a new 50 ml falcon tube.
6. Wash the lymphocytes 2 x with PBS at 1500 rpm, at RT, 10 min, with brake on in Sorvall table centrifuge.
7. Use CLL-P100- protocol to purify BCL2 containing membranes.
*PBS: 25 x: 5g Kcl
5g KH2PO4
29g Na2HPO4 x 2 H2O
200g NaCl
dilute to 1x with MQ water and autoclave before use.
**Ficoll-Paque: purchase from Pharmacia LKB Code No: 17-0840-03
