Purpose: To isolate Peripheral Blood Mononuclear Cells (PBMCs) from a blood sample.

Principle: Ficoll is a hydrophilic polysaccharide solution with a density of approximately 1.077 g/mL that is layered beneath the blood sample and centrifuged. It is commonly used to separate blood components based on their density, resulting in three distinct layers after centrifugation: plasma (top), PBMCs (interface), and red blood cells (bottom). During centrifugation, cells experience a centrifugal force that causes them to migrate until they reach a region where their density equals that of the surrounding solution. Since red blood cells and granulocytes are denser than Ficoll, they pass through it and settle at the bottom. Plasma, which is the least dense, moves to the top, while PBMCs form a layer between the plasma and Ficoll. This layer is known as the buffy coat.

Information on the Centrifuge Used Swing-bucket centrifuges hang vertically at rest but swing horizontally during centrifugation. The tubes swing out perpendicular to the axis of rotation, allowing the centrifugal force to act directly down the length of the tube. This keeps the layers undisturbed and perfectly horizontal, making this type of centrifuge ideal for separating density layers. In contrast, fixed-angle rotor centrifuges hold the tubes at a constant angle relative to the axis of rotation. As a result, the centrifugal force acts at an angle, causing components to move diagonally and form pellets along the side and bottom of the tube. Thus, swing-bucket centrifuges are ideal for PBMC isolation.

Protocol

  1. Dilute the blood based on its thickness (usually a 1:1 ratio of blood to saline).
  2. Layer Ficoll (1:3 ratio of Ficoll to blood) into a 15 mL tube without mixing the two layers.
  3. Centrifuge at 1500 rpm for 20 minutes.
  4. Discard the plasma layer and collect the buffy coat into a new 15 mL tube.
  5. Dilute with saline to a total volume of 10 mL.
  6. Centrifuge for 10 minutes at 1500 rpm.
  7. Discard the supernatant and resuspend the pellet in saline or DMEM.
  8. Count the cells using crystal violet dye.