Effects of different density gradient separation techniques on neutrophil function

Abstract

Investigation of the pathogenesis of inflammatory diseases has involved assessment of polymorphonuclear leukocyte (PMN) activity and a variety of techniques for separating PMNs from whole blood have been described. In this study the effects of Percoll gradient, Ficoll-Hypaque/Dextran sedimentation (FH/DS) and Mono-Poly Resolving Medium (M-PRM) on activation and function of PMNs were compared. All three separation techniques gave similar cell yield and purity. The mean (±SEM) percentage of cells demonstrating pseudo-podia formation for Percoll, FH/DS and M-PRM were 39 ± 9, 57 ± 6 and 63 ± 5, respectively, while superoxide release from resting cells was 1.9 ±0.9, 7.2 ±3.5 and 11±4.8pmols per 10⁶ cells min^-1, respectively, indicating that activation of cells during separation may be less with Percoll compared to the other methods. The functional capacity of the cells to respond to a stimulus was similar for all methods as indicated by similar EC₅₀ values for chemotaxis to zymosan-activated serum and similar superoxide production induced by tet-radecanoyl phorbol acetate. All three separation techniques produce functionally active PMNs of high purity but the use of Percoll gradients may be preferable when a quick method of separation which causes minimum pre-acti-vation of PMNs is required.

Key Words:

• chemotaxis
• neutrophil activation
• oxidants