Density separation of erythrocyte sub-populations

Solutions Hepes buffer. Hepes (20 mM), sodium chloride (135 mM), potassium chloride (5 mM), magnesium chloride (1 mM), disodium hydrogen phosphate (1 mM), glucose (5 mM), pH 7.40, osmolarity 300 mOsm/kg H20. 

Percoll-diatrizoate solution-. 10.0 ml of sodium diatrizoate in water (50%, w/v) are mixed with 45.0 ml Percoll. Hepes buffer (10.0 ml) is added to 49.5 ml of the above mixture and the osmolarity is adjusted 

Procedure Packed erythrocytes, washed twice in Hepes buffer, are resuspended in an equal volume of the same buffer. The erythrocyte suspension (2 ml) is carefully layered over the Percoll-diatrizoate solution (2 ml) in a glass centrifuge tube with an internal diameter of 10 mm. After centrifugation at 400 x g for 20 min at room temperature, erythrocytes with normal density are carefully removed from the surface of the Percoll-diatrizoate cushion using a pasteur pipette. This fraction is referred to as the normal density fraction. Erythrocytes with elevated density, which had pelleted below the Percoll-diatrizoate, can then be removed in a similar manner. This latter fraction is referred to as the dense fraction. Finally, each fraction of erythrocytes is washed twice in 10 vol. of Hepes buffer at 4°C. 

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