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Activation of Nuclear Factor Kappa B (NF-κB) Assayed by Laser Scanning Cytometry (LSC)

机译：激光扫描细胞仪（LSC）检测核因子κB（NF-κB）的活化

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Nuclear factor kappa B (NF-κB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-κB remains in the cytoplasm sequestered through interaction with IκB protein. Rapid translocation of NF-κB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-κB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor α (TNF-α) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-κB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counter-stained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-κB led to a rapid increase in NF-κB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (FC), which was reflected by an increase in FN/FC ratio. This rapid assay of NF-κB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-κB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.
机译：核因子κB（NF-κB）/ rel是参与调节各种免疫和炎症反应的普遍存在的转录激活因子家族。它还在控制细胞生长和凋亡中起作用。 NF-κB以其非活性形式保留在通过与IκB蛋白相互作用而被隔离的细胞质中。响应细胞外信号而发生的NF-κB从细胞质快速转移到细胞核被认为是其激活的标志性特征。目前，通过激光扫描细胞仪测量了HL-60，U-937和Jurkat白血病细胞以及肿瘤坏死因子α（TNF-α）或佛波醇肉豆蔻酸乙酸酯（PMA）诱导的人成纤维细胞中NF-κB的移位（ LSC）。 NF-κB用FITC标记的抗体进行了免疫细胞化学检测，并通过测量整合在细胞核上的绿色荧光来监测其在细胞核中的存在，并用碘化丙锭（PI）和细胞质。 NF-κB的活化导致在细胞核（FN）上测得的NF-κB相关荧光迅速增加，而在细胞质（FC）上的荧光下降，这由FN / FC比的增加反映出来。可以将这种NF-κB活化的快速测定与活化细胞的形态学鉴定或其免疫表型结合起来。 NF-κB表达与细胞DNA含量的双变量分析使其与细胞周期位置相关联成为可能。所描述的方法有潜力用作功能测定，以监测其他转录激活因子（例如p53肿瘤抑制蛋白或信号转导分子）的细胞内易位。

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