The entire spectrum of cell types present in blood can be resolved on preformed gradients of Percoll. The method described by Pertoft et al. (55) (Fig 19) utilizes both rate zonal (separation by size) and isopycnic (separation by density) techniques. Diluted blood was layered on top of a preformed self-generated gradient and centrifuged for 5 min at 400 × g, during which time the thrombocytes or platelets (which are appreciably smaller than the other cells present) did not penetrate into the gradient.
The plasma layer containing the thrombocytes was removed and replaced by saline, and centrifugation was continued at 800 × g for 15 min, resulting in isopycnic banding of mononuclear cells (lymphocytes and monocytes), polymorphonuclear cells and erythrocytes. The position and densities of the banded cells were monitored using Density Marker Beads in an identical gradient contained in a second centrifuge tube.
Although the above method demonstrates the utility of Percoll for fractionating whole blood, most blood cells can be appreciably enriched using a simple step gradient. A simple step gradient often gives acceptable yields and purity for downstream processing. The Application Tables below contain a number of examples of purification of blood cells and other cell types using different types of Percoll gradients.
Figure 19.Separation of human blood cells in a gradient of Percoll. The tubes were filled with 10 ml of 70% (v/v) Percoll in 0.15 M NaCl (p=1.086 g/ml), and the gradient performed by spinning in a 14° angle rotor at 20 000 × g for 15 min. Two ml of gradient material was removed from the bottom of the tube using a syringe, and 1 ml of heparinized blood diluted with 1 ml of 0.15 M NaCl was layered on top of the gradient. Centrifugation was carried out as indicated. Densities were monitored using Density Marker Beads. MNC = Mononuclear cells, PMNC = Polymorphonuclear cells, RBC = Red blood cells (55, reproduced by kind permission of the authors and publisher).
The following tables were compiled to assist the researcher in selecting references most likely to contain relevant information regarding use of Percoll for a particular cell or tissue type.