Kolber M A, Quinones R R, Gress R E, Henkart P A
Immunology Branch, National Cancer Institute, Bethesda, MD 20892.
J Immunol Methods. 1988 Apr 6;108(1-2):255-64. doi: 10.1016/0022-1759(88)90427-9.
In order to utilize a newly available scanning microfluorimeter for lymphocyte-mediated cytotoxicity assays, a number of commercially available fluorescent dyes were compared for their suitability as target cell markers. One of them, bis-carboxyethyl-carboxyfluorescein (BCEFCF), was useful for assays with about 10(4) target cells and showed substantially less spontaneous leakage than other fluorescein derivatives, while still leaking more rapidly than 51Cr. For short cytotoxicity incubations (less than 2 h) with cytotoxic T lymphocytes (CTL), the corrected percentage BCECF release into the supernatant parallels that of 51Cr. For 4 h assays cytotoxicity could be quantitated by measuring the BCECF retained by target cells. Using human CTL and natural killer (NK) cells as effectors, with a variety of lymphoid cells and fibroblasts as targets in 4 h assays, the BCECF retention technique was found to give cytotoxicity values comparable to the 51Cr release assay. Cytotoxicity assays measuring BCECF fluorescence in microtiter wells with the scanning microfluorimeter offer advantages of safety, economy, and processing time compared with the 51Cr release assay.
为了将新获得的扫描式显微荧光计用于淋巴细胞介导的细胞毒性测定，对多种市售荧光染料作为靶细胞标记物的适用性进行了比较。其中一种双羧乙基羧基荧光素（BCEFCF），适用于约10⁴个靶细胞的测定，与其他荧光素衍生物相比，其自发泄漏明显较少，不过仍比⁵¹Cr泄漏得更快。对于与细胞毒性T淋巴细胞（CTL）进行的短时间细胞毒性孵育（少于2小时），释放到上清液中的经校正的BCECF百分比与⁵¹Cr的情况相似。在4小时的测定中，细胞毒性可以通过测量靶细胞保留的BCECF来定量。以人CTL和自然杀伤（NK）细胞作为效应细胞，在4小时的测定中以多种淋巴细胞和成纤维细胞作为靶细胞，发现BCECF保留技术给出的细胞毒性值与⁵¹Cr释放测定法相当。与⁵¹Cr释放测定法相比，用扫描式显微荧光计测量微量滴定孔中BCECF荧光的细胞毒性测定法具有安全、经济和处理时间短的优点。
