Question

How do Ficoll-density gradients separate white blood cells?

Answer 1

Defibrinated or anticoagulant-treated blood is diluted with an equal volume of a balanced salt solution, layered carefully over Ficoll-Paque product (without intermixing) and centrifuged for 30 to 40 min. Differential migration of cells during centrifugation results in the formation of layers containing different cell types:

• The bottom layer contains erythrocytes, which have been aggregated by Ficoll PM400 and therefore sediment completely in the Ficoll-Paque density gradient media layer.
• The layer immediately above the erythrocyte layer contains mostly granulocytes, which at the osmotic pressure of the Ficoll-Paque media solution, attain a density high enough to migrate through the Ficoll Paque media layer.
• At the interface between the plasma and the Ficoll-Paque layer, mononuclear cells are found together with other slowly sedimenting particles (e.g., platelets) with low density.

Lymphocytes, monocytes, and platelets are not dense enough to penetrate into the Ficoll-Paque media layers having densities of 1.077 and 1.084 g/ml. These cells therefore collect as a concentrated band at the interface between the original blood sample and the Ficoll-Paque products having densities of 1.077 and 1.084 g/ml.