Concurrent isolation of granulocytes and lymphocytes with unaltered permeability, energy state, and metabolic capacity in vitro.

Although numerous procedures have been described for the isolation of either granulocytes or lymphocytes, few focused on the concurrent separation of these cells from the same sample of blood. Those reported (e.g., (1)) are based on the use of discontinuous gradients of Ficoll-Hypaque (sodium diatrizoate), a method introduced by Boyum (2). However, a common disadvantage of these procedures is the substantial contamination of the isolated granulocytes and lymphocytes by erythrocytes and platelets. Our aim, to extend the investigation of the mechanisms of cellular drug transport in leukocytes (3, 4) to granulocytes and lymphocytes, required the availability of corresponding cellular fractions of high purity in which the cells fulfill stringent criteria of viability. The presence of an intact plasma membrane during experiments focusing on transport is an essential prerequisite in utilizing cells in such studies. Plasma membrane integrity has to be evaluated considering stringent criteria, sensitive enough to detect even slight perturbations of the plasma membrane (5). Furthermore, the isolated cells have to fall the aforesaid and additional criteria of viability over a prolonged time, to allow their use in experiments in vitro. We have previously described a method for the isolation of human and rat leukocytes, erythrocytes, and platelets which fulfilled the above outlined requirements (6). This paper describes a procedure for the concurrent isolation and separation of human granulocytes and lymphocytes, focusing on high purity and long-term viability in vitro.