Radosević K, Garritsen H S, Van Graft M, De Grooth B G, Greve J
Department of Applied Physics, Twente University of Technology, Enschede, The Netherlands.
J Immunol Methods. 1990 Dec 31;135(1-2):81-9. doi: 10.1016/0022-1759(90)90259-x.
A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.
本文描述了一种用于测定人自然杀伤细胞细胞毒性活性的新型、简单且灵敏的流式细胞术检测方法。该检测方法基于两种荧光染料的使用。在与效应细胞孵育之前，用一种荧光染料（十八烷基胺 - 异硫氰酸荧光素，F - 18）对靶细胞群体进行染色。即使靶细胞被杀死，F - 18仍保留在其细胞膜中，从而使得效应细胞和靶细胞能够清晰分离。在效应细胞和靶细胞孵育后，用第二种荧光染料（碘化丙啶）进行染色来确定死亡细胞。F - 18无毒，不会降低人自然杀伤细胞的细胞毒性活性。它也很稳定（在37℃下至少4小时内，标记细胞与未标记细胞之间的交换可忽略不计），并且会保留在被杀死细胞的细胞膜中。即使在37℃下将靶细胞和效应细胞孵育4小时并使用高效应细胞 - 靶细胞比例（75:1）后，也能清晰区分未标记的效应细胞和标记的靶细胞。该方法与51Cr释放试验具有良好的相关性。本文还介绍了使用全血而非分离淋巴细胞进行流式细胞术细胞毒性检测的潜在应用。
