[Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b].

A Andoh, Y Fujiyama, S Hirotani, K Hodohara, T Bamba, S Hosoda
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Abstract

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.

[使用含C3b的致敏荧光微球进行免疫吞噬作用的流式细胞分析]。
我们用IgG或IgG致敏的荧光乳胶珠分析纯化的人单核细胞的吞噬活性。流式细胞术检测C3。为了制备IgG致敏乳胶珠(BA),将bsa包被的乳胶珠(B)与稀释的兔抗bsa IgG孵育。为了结合补体成分,BA与用K-76单羧酸(K-76COOH)预处理的全血清孵育。K-76COOH抑制因子I和C5的活性,导致C1, C4b, C2a, C3b沉积在BA (BAC)上。以吞噬率和吞噬指数(PI)评价吞噬活性。为了消除未被吞噬的乳胶珠的影响,从37℃中减去4℃时的数据。60 min的吞噬率分别为b5.0%、BA 18.3%和BAC 57.5%, PI(摄入乳胶珠/100细胞)分别为b7.9、BA 36.8和BAC 152.7。此外,K-76COOH对IgG具有剂量依赖性抑制作用。C3介导的吞噬作用。对BAC和BA的抑制模式比较表明,K-76COOH直接抑制C3。C3-receptor绑定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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