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文章介绍流式细胞仪(FC)和荧光原位杂交技术(FISH)联合运用,作为定量检测选择细胞群中特异性细胞因子mRNA水平的敏感方法.为了检测小鼠体内诱导的IL-1αmRNA,首先于BALB/C小鼠腹膜内注入20μgLPS,3小时后收集脾细胞和硫基乙醇诱导的腹腔渗出细胞.每组细胞标记大鼠抗小鼠巨噬细胞抗体(MAC)或IA~d.RNA-RNA FISH测定,将悬浮细胞与生物素化的有意义IL-1α探针,反义IL-1α和反义IL-2探针共同孵育37℃17小时.杂交细胞用RNA酶处理,异硫氰酸荧光素
This article describes the combination of flow cytometry (FC) and fluorescence in situ hybridization (FISH) as a sensitive method for quantitative detection of specific cytokine mRNA levels in selected cell populations. To detect IL-1α mRNA induced in mice, first 20 μg of LPS was intraperitoneally injected into BALB/C mice, and spleen cells and thioethanol-induced peritoneal exudate cells were collected 3 hours later. Each group of cells was labeled with rat anti-mouse macrophage antibody (MAC) or IA~d.RNA. -RNA FISH assay, suspension cells were incubated with biotinylated IL-1α probe of interest, antisense IL-1α and antisense IL-2 probe at 37°C for 17 hours. Hybrid cells treated with RNase, isothio Cyanine fluorescein