Isolation of polymorphonuclear leukocytes (granulocytes) from rodent and rabbit blood

A common approach to the problem of isolating rodent and rabbit polymorphonuclear leukocytes (PMNs) was to use a density barrier of Nycoprep™ 1.077A (ρ = 1.077 g/ml, osmolality = 265 mOsm). 

The method was first published by Bøyum et al [1]. This customized medium separated the mononuclear cells (MCs), which band at the interface, from the PMNs and the erythrocytes, which sediment through the barrier to form a pellet. To retrieve the PMNs, the erythrocytes may be selectively lysed in isotonic ammonium chloride solution or ice-cold distilled water.

 An alternative approach is to remove the erythrocytes from whole blood first by aggregation with dextran [2-4], methylcellulose [5,6] hetastarch [7] or Plasmagel [8,9] and then layer the resulting leukocyte-rich plasma (LRP) over the Nycoprep™ 1.077A, so that the PMN pellet contains only a very small percentage of residual erythrocytes. Nycoprep™ 1.

077A is no longer commercially available but an iodixanol solution of the same density and osmolality can be easily prepared from OptiPrep™ and it is this method that is described in Section 2.