Boogaerts M A, Vercelotti G, Roelant C, Malbrain S, Verwilghen R L, Jacob H S
Scand J Haematol. 1986 Sep;37(3):229-36. doi: 10.1111/j.1600-0609.1986.tb02302.x.
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.
用于体外研究的粒细胞制备“标准”技术采用葡聚糖去除红细胞以及Ficoll-Hypaque梯度离心法来提高粒细胞纯度。该过程冗长，在我们操作中约需150分钟，且所获得的粒细胞被血小板严重污染（约每中性粒细胞含5个血小板）。我们报告一种技术，用羟乙基淀粉替代葡聚糖，用Percoll替代Ficoll-Hypaque。制备时间减少约40%，血小板污染减少超过80%。如此制备的粒细胞在代谢功能（产氧、化学发光、磷酸戊糖途径最大值）方面，以及在运动性/吞噬作用测定中，与“标准”制备的粒细胞表现相同。然而，发现血小板在粒细胞聚集反应中有增强作用及其对细胞毒性的介导作用。与Percoll制备的细胞（血小板含量低）相比，Ficoll-Hypaque纯化的细胞（富含血小板）与FMLP或活化补体凝集素的聚集程度明显更高，并且过度杀伤51Cr标记的靶细胞。将纯化的血小板或血小板释放上清液重新添加到后者中，可重现使用“标准”制备方法时的结果。
