Leucocytes/PBMCs isolation from whole blood

Procedures

Preparation

•   Separating solution should be at RT
•   Pipette 15ml separating solution into Leucosep-tube
•   Centrifuge tube with separating solution 30sec 1000g at RT
•   Dilute 10ml blood 1:2 with 0.9 % sodium chloride solution, final volume 30ml

Isolation

•   Add carefully blood solution to the Leucosep tube (separating solution is already inside)
•   Centrifuge 15min 800g at RT without brake!
•   Pipette intermediate phase (leucocytes/PBMCs) into a new 15ml tube (lower phase (cell pellet): erythrocytes/granulocytes, middle phase: leucocytes/PBMCs (above the filter and separating solution), upper phase: plasma
•   Wash cells with 10ml PBS 10min 250g at RT, remove supernatant
•   Resuspend cell pellet with 5ml PBS, centrifuge 10min 250g at RT, remove supernatant
•   Repeat step 5

Materials

HRTC-medium
Leucosep-tube, sterile, 50ml	Greiner Bio-One #227290
Biocoll separating solution	Biochrom AG #L6115
0.9 % sodium chloride solution