In-Cell Western assay: a new approach to visualize tissue factor in human monocytes
In-Cell Western assay: a new approach to visualize tissue factor in human monocytes
复制标题
DOI：
10.1111/j.1538-7836.2005.01781.x
复制
发表时间：
2006-03-01
影响因子：
10.4
通讯作者：
Osterud, B
中科院分区：
文献类型：
--
作者：
Egorina, EM;Sovershaev, MA;Osterud, B
背景：组织因子（TF）是启动外源性止血途径所必需的膜蛋白。对信托基金监管的准确理解仍然有限，并取决于方法工具的可用性。在这里，我们描述了一种新的方法来评估TF量在人单核细胞（MNCs）通过使用全血实验条件。目的：为了研究TF抗原在人的MNCs的水平，我们应用了定量免疫染色技术-在细胞蛋白质（ICW）测定使用奥德赛红外成像系统。方法和结果：在静息或脂多糖（LPS）刺激的人MNCs中进行TF的ICW测定。测试了几种样品制备条件，即在免疫染色之前接种MNC、多聚甲醛固定，并在测定中使用足够的细胞数量。通过使用重组人TF标准品，首次可以测量LPS刺激的MNC中的TF量，分别为0.09 +/- 0.02 ng和0.43 +/- 0.15 ng 10（-6）细胞表面和总TF。然而，TF在静息MNCs中的浓度低于检测限。结论：一种新的TF ICW测定是一种可重复的、节省时间和成本的方法，可用于人类止血生理学和病理生理学领域的研究。
Background: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. Aim: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique - in-cell Western (ICW) assay using an Odyssey Infrared Imaging System. Methods and results: The ICW assay of TF in resting or lipopolysaccharide (LPS)-stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS-stimulated MNCs as 0.09 +/- 0.02 ng and 0.43 +/- 0.15 ng 10(-6) cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. Conclusions: A novel TF ICW assay is a reproducible, time- and cost-saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.