Datta Subhash C, Opp Mark R
Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48109, USA.
J Neurosci Methods. 2008 Oct 30;175(1):119-24. doi: 10.1016/j.jneumeth.2008.08.007. Epub 2008 Aug 14.
Methods to determine cytokine protein content in samples of interest, such as enzyme-linked immunosorbent assay (ELISA), are often labor-intensive and costly. Furthermore, because ELISA requires relatively large sample volumes and protein concentrations, it is difficult using this technique to determine protein content for multiple cytokines from individual samples. Recently, Luminex has developed an open source hardware platform combining flow cytometry- and bead-based antibody capture that is capable of detecting multiple analytes from a single sample. In the present study we employed the Luminex 200 platform to determine the cytokine protein content in discrete brain regions of C57BL/6J mice. In spike-and-recovery experiments, known concentrations of murine recombinant interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)alpha were added either singly or as a mixture of all three to whole brain homogenates containing known quantities of total protein. Spiked samples were assayed for either a single cytokine or for multiple cytokines using 1-plex or 3-plex assay kits, respectively. In whole mouse brain homogenate we recovered between 81% and 103% of the recombinant cytokines. We then injected C57BL/6J mice intraperitoneally with bacterial lipopolysaccharide (LPS) and sacrificed them 4h later. We detected in samples taken from LPS-stimulated mice 4- to 870-fold increases in serum or spleen cytokine protein, and 1.5- to 16-fold increases in cytokine protein in discrete brain regions, relative to protein content in samples obtained from vehicle-treated animals. These results indicate that multiple cytokines may be reliably assayed from discrete regions of mouse brain using a single sample.
用于测定感兴趣样本中细胞因子蛋白含量的方法,如酶联免疫吸附测定(ELISA),通常 labor-intensive 且成本高昂。此外,由于 ELISA 需要相对大量的样本体积和蛋白浓度,使用该技术很难从单个样本中测定多种细胞因子的蛋白含量。最近,Luminex 开发了一种开源硬件平台,该平台结合了流式细胞术和基于微珠的抗体捕获技术,能够从单个样本中检测多种分析物。在本研究中,我们使用 Luminex 200 平台来测定 C57BL/6J 小鼠离散脑区中的细胞因子蛋白含量。在加标回收实验中,将已知浓度的小鼠重组白细胞介素(IL)-1β、IL-6 和肿瘤坏死因子(TNF)α 单独或作为三者的混合物添加到含有已知总量蛋白的全脑匀浆中。加标样本分别使用 1 重分析试剂盒或 3 重分析试剂盒检测单一细胞因子或多种细胞因子。在全小鼠脑匀浆中,我们回收了 81%至 103%的重组细胞因子。然后,我们给 C57BL/6J 小鼠腹腔注射细菌脂多糖(LPS),并在 4 小时后将其处死。相对于从接受溶剂处理动物获得的样本中的蛋白含量,我们在从 LPS 刺激的小鼠采集的样本中检测到血清或脾脏细胞因子蛋白增加了 4 至 870 倍,离散脑区中的细胞因子蛋白增加了 1.5 至 16 倍。这些结果表明,使用单个样本可以可靠地从小鼠脑的离散区域中检测多种细胞因子。