Sellers J R, Cook S, Goldmacher V S
ImmunoGen, Inc., Cambridge, MA 02139-4239.
J Immunol Methods. 1994 Jun 24;172(2):255-64. doi: 10.1016/0022-1759(94)90112-0.
A cytotoxicity assay has been developed based on the measurement of the proliferative activity of surviving cells as quantified by a cell-incorporated fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF proliferative assay is fast (the results are obtained within 3-4 days depending on the cell line), accurate, not labor-intensive, does not require the use of radioisotopes or toxic compounds, and is amenable to automation. The BCECF proliferative assay was compared with two other indirect cytotoxicity tests, a trypan blue exclusion test and a BCECF viability test. Neither of these two latter assays reflected in any way the killing of cells by ricin. In contrast, using the BCECF proliferation assay, an optimal period of cell culturing after exposure to a toxin could be found so that the cytotoxicity values produced agreed with the surviving fractions of cells measured in a direct cytotoxicity assay. Under non-optimal conditions, the assay reflected the cell kill only qualitatively. Although it is common practice to conduct indirect cytotoxicity tests without validating them with a direct assay, our experiments demonstrate that the values obtained in such non-optimized indirect cytotoxicity tests may not parallel the cell kill and may, therefore, be meaningless.
基于对存活细胞增殖活性的测量，开发了一种细胞毒性测定方法，该活性通过细胞内掺入的荧光染料2',7'-双-(2-羧乙基)-5(6)-羧基荧光素（BCECF）进行定量。BCECF增殖测定法速度快（根据细胞系不同，3 - 4天内可获得结果）、准确、不耗费人力、无需使用放射性同位素或有毒化合物，并且适合自动化操作。将BCECF增殖测定法与另外两种间接细胞毒性试验——台盼蓝排斥试验和BCECF活力试验进行了比较。后两种试验均未以任何方式反映蓖麻毒素对细胞的杀伤作用。相比之下，使用BCECF增殖测定法，可以找到暴露于毒素后细胞培养的最佳时期，从而使产生的细胞毒性值与直接细胞毒性测定中测得的细胞存活分数一致。在非最佳条件下，该测定法仅定性地反映细胞杀伤情况。尽管在不通过直接测定进行验证的情况下进行间接细胞毒性试验是常见做法，但我们的实验表明，在这种未优化的间接细胞毒性试验中获得的值可能与细胞杀伤情况不平行，因此可能毫无意义。
