基于细胞的流式细胞术检测以测量细胞毒性活性。
Cell-based flow cytometry assay to measure cytotoxic activity.
作者信息
Noto Alessandra, Ngauv Pearline, Trautmann Lydie
机构信息
Vaccine and Gene Therapy Institute of Florida.
Vaccine and Gene Therapy Institute of Florida;
出版信息
J Vis Exp. 2013 Dec 17(82):e51105. doi: 10.3791/51105.
Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.
CD8+ T细胞的细胞溶解活性很少被评估。我们在此描述一种新的基于细胞的检测方法，用于测量抗原特异性CD8+ T细胞杀伤负载其同源肽的CD4+ T细胞的能力。将靶CD4+ T细胞分为两个群体，用两种不同浓度的羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）进行标记。一个群体用感兴趣的肽进行脉冲处理（CFSE低），而另一个群体不进行脉冲处理（CFSE高）。将脉冲处理和未脉冲处理的CD4+ T细胞以相等比例混合，并与数量不断增加的纯化CD8+ T细胞一起孵育。在与含有通过肽/MHC I四聚体染色检测到的抗原特异性效应CD8+ T细胞的CD8+ T细胞共培养后，通过流式细胞术分析自体靶CD4+ T细胞的特异性杀伤情况。在不同效应细胞与靶细胞比例下测量靶CD4+ T细胞的特异性裂解，可计算裂解单位，即LU₃₀/10⁶个细胞。这种简单直接的检测方法能够准确测量CD8+ T细胞杀伤靶CD4+ T细胞的内在能力。
Zotero 插件