Nakagawa Yohko, Watari Eiji, Shimizu Masumi, Takahashi Hidemi
Department of Microbiology and Immunology, Nippon Medical School, Sendagi, Bunkyo-ku Tokyo, JAPAN.
Biomed Res. 2011 Apr;32(2):159-66. doi: 10.2220/biomedres.32.159.
Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ( 51)Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical (51)Cr-release assay.
使用基于荧光的染料5-（和6-）羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）在体内检测细胞毒性T淋巴细胞（CTL）的方法已经建立并得到广泛应用。基于这一经验，我们将其应用于体外检测系统，建立了一种简单、高度灵敏的流式细胞术检测CTL活性的方法。在我们的检测中，通过在单色直方图分析中敏感靶细胞数量的减少来检测CTL的特异性活性。通过使用这种检测方法，我们可以确定表位肽内单个氨基酸取代对细胞毒性活性的影响。在与效应CTL孵育后，通过用过量的EDTA和胰蛋白酶试剂处理，贴壁细胞也可作为该检测中的靶细胞。此外，当使用荧光校准微球作为对照时，我们可以确定CTL对肿瘤细胞的细胞毒性。我们的检测结果与传统的（51）铬释放检测结果几乎一致。因为我们的检测仅使用一种稳定的非放射性试剂CFSE，所以该检测安全、廉价且极其简便。这些结果表明，这种新的检测方法（FACS-CTL检测）将是经典（51）铬释放检测的一个完全可接受的替代方法。
