Platelets augment granulocyte aggregation and cytotoxicity: uncovering of their effects by improved cell separation techniques using Percoll gradients.
The 'standard' technique of granulocyte preparation for in vitro studies uses dextran removal of erythrocytes and Ficoll-Hypaque gradient centrifugation to increase granulocyte purity. The procedure is lengthy, approximately 150 min in our hands, and provides granulocytes significantly contaminated with platelets (approx. 5 platelets/PMN). We report a technique that replaces dextran with hydroxy-ethylstarch and Ficoll-Hypaque with Percoll. Preparation time is reduced by approximately 40% and platelet contamination by more than 80%. Granulocytes, so prepared, function metabolically (O2-generation, chemiluminescence, HMP-shunt maxima) and, in motility/phagocytosis assays, identically to 'standard' preparations. However, an augmentatory effect of platelets in granulocyte aggregation responses and their mediation of cytotoxicity is uncovered. Ficoll-Hypaque purified cells (platelet-rich) aggregate to a significantly greater degree with FMLP or activated complement lectins and excessively kill 51Cr-labelled target cells when compared to Percoll-preparations (platelet-poor). Re-addition of purified platelets or of platelet release supernatants to the latter reproduces results using the 'standard' preparations.
D001792:Blood Platelets / Q000502:physiology*; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D003602:Cytotoxicity, Immunologic; D003973:Diatrizoate; D005362:Ficoll; D006098:Granulocytes / Q000502:physiology*; D006801:Humans; D008163:Luminescent Measurements; D010587:Phagocytosis; D011205:Povidone; D012822:Silicon Dioxide; D013481:Superoxides / Q000096:biosynthesis
D013481:Superoxides; D003973:Diatrizoate; D005362:Ficoll; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone