Oben J A, Foreman J C
Department of Pharmacology, University College London, U.K.
J Immunol Methods. 1988 Aug 9;112(1):99-103. doi: 10.1016/0022-1759(88)90039-7.
In general the in vitro assays of phagocytosis rely on microscope counting or radioisotopic detection of ingested particles or microbiological counting of non-ingested bacteria. A very simple, rapid, highly quantitative method using fluorescein-labelled bacteria was described by Vray et al. (Scand. J. Immunol. (1980) 11, 147) for non-human phagocytes. We report here a modification of this method to increase its sensitivity, to make it more suitable for pharmacological studies. We also provide detailed experimental parameters for its use with human phagocytes. A suspension of fluorescein-labelled bacteria is incubated with human phagocytes; after incubation at 37 degrees C, the reaction is terminated with ice-cold Tyrode buffer solution, and the non-ingested bacteria are removed by lysis with lysozyme and the resultant cell suspension treated with the detergent TX-100. The fluorescence of the suspension is then measured. The modified method is sufficiently sensitive to permit the detection of bi-directional effects on phagocytosis of a known modulator of human phagocyte function.
一般来说，吞噬作用的体外测定依赖于显微镜计数、对摄入颗粒的放射性同位素检测或对未摄入细菌的微生物计数。Vray等人（《斯堪的纳维亚免疫学杂志》（1980年）11卷，第147页）描述了一种使用荧光素标记细菌的非常简单、快速、高度定量的方法，用于非人类吞噬细胞。我们在此报告对该方法的改进，以提高其灵敏度，使其更适合药理学研究。我们还提供了将其用于人类吞噬细胞的详细实验参数。将荧光素标记细菌的悬浮液与人类吞噬细胞一起孵育；在37℃孵育后，用冰冷的台氏缓冲液终止反应，并用溶菌酶裂解去除未摄入的细菌，并用去污剂TX - 100处理所得的细胞悬浮液。然后测量悬浮液的荧光。改进后的方法灵敏度足够高，能够检测对人类吞噬细胞功能的已知调节剂的吞噬作用的双向影响。
