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