Brown A R, Fishman M
Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Cell Immunol. 1990 Oct 15;130(2):352-63. doi: 10.1016/0008-8749(90)90278-y.
Immunocytochemical staining procedures were combined with computer-aided image analysis to quantitate the relative intracellular production of tumor necrosis factor-alpha (TNF) within individual macrophages. Optimal conditions for time and methods for the activation of TNF production, fixation of cells for optimal immunocytochemical staining, and image analysis methods were determined. Thioglycolate elicited peritoneal macrophages were readily activated to significantly increased levels of intracellular TNF, as early as 1 hr after activation with lypopolysaccharide (LPS) + interferon-gamma: maximum intracellular TNF was evident after 2-3 hr. Both LPS and interferon-gamma was necessary to increase intracellular TNF. Normal alveolar macrophages also readily produced increased intracellular TNF, but normal peritoneal and splenic macrophages were poorly activated to TNF production. Acid stripping of receptor bound TNF allowed discrimination between intracellular TNF/integral membrane TNF, and TNF-receptor-bound TNF. Results stress the importance of studying these TNF forms early after activation. Applications for TNF quantitation by these means are discussed.
免疫细胞化学染色程序与计算机辅助图像分析相结合，以定量单个巨噬细胞内肿瘤坏死因子-α（TNF）的相对细胞内产生量。确定了激活TNF产生的时间和方法、固定细胞以进行最佳免疫细胞化学染色以及图像分析方法的最佳条件。早在用脂多糖（LPS）+γ干扰素激活后1小时，巯基乙酸诱导的腹腔巨噬细胞就很容易被激活，细胞内TNF水平显著升高：2-3小时后细胞内TNF达到最大值。LPS和γ干扰素都是增加细胞内TNF所必需的。正常肺泡巨噬细胞也很容易产生细胞内TNF增加，但正常腹腔和脾巨噬细胞对TNF产生的激活较差。受体结合的TNF的酸剥离允许区分细胞内TNF/整合膜TNF和TNF受体结合的TNF。结果强调了在激活后早期研究这些TNF形式的重要性。讨论了通过这些方法进行TNF定量的应用。
