Moghe:PBMC

Isolation of mononuclear cells from peripheral blood Buffy Coats

Materials

• 1.077g/mL Ficoll Paque Plus (GE healthcare) at room temperature
• Sterile 50mL conical tubes
• Sterile PBS
• RPMI 1640 (10% FBS, 1%pen/strep)
• Buffy coat(s)
  • Buffy coats are isolated from whole blood at the blood bank by an initial centrifugation without a density gradient. This initial spin concentrates red cells at the bottom and plasma on top, the buffy coat forms the interface and contains most of the leukocytes from the whole blood (although it still looks like blood). Each unit (~550mL) of whole blood yields a ~45mL buffy coat, which contains ~200-800*10⁶ mononuclear cells.

All work should be done inside a cell culture hood

Procedure

• Keep units of blood separate and record lot information
• Open the bag(s) of buffy coat with a razor or scalpel and transfer each into 50mL tube
• Add 15mL ficoll to 2 50mL tubes for each buffy coat.
• Layer half of the buffy coat on top of ficoll carefully with a serological pipet
• Centrifuge at room temp – 400g for 30 min with 0 acceleration/deceleration
• Asipirate off top layer containing residual plasma
• Collect 2nd layer mononuclear cells without taking from the ficoll layer below and transfer to new 50mL tube
• Add 40mL PBS to wash the cells mixing thoroughly
• Centrifuge for 15 min at 400g – room temp, full acceleration/deceleration
• Aspirate PBS and add 1mL ACK buffer
• After 1 minute, add 40mL PBS
• Centrifuge for 10 min at 300g – room temp, full acceleration/deceleration
• Aspirate PBS and add 35mL PBS
• Centrifuge for 10 min at 200g – room temp, full acceleration/deceleration
• Aspirate PBS and resuspend each pellet in 5mL RPMI 1640 (10% FBS)
• Combine pair of resuspended cells (keep different buffy coats separate) and count