A NOVEL ONE-STEP, HIGHLY SENSITIVE FLUOROMETRIC ASSAY TO EVALUATE CELL-MEDIATED CYTOTOXICITY

In this study, a fluorometric method using alamarBlue has been develop ed for detecting cell-mediated cytotoxicity in vitro. AlamarBlue is a non-toxic metabolic indicator of viable cells that becomes fluorescent upon mitochondrial reduction. Specific lysis of targets by effector c ells is quantified by comparing the total number of viable cells in we lls containing effector and targets together, with wells where target and effector cells were separately seeded. Cell-mediated cytotoxic act ivity by alloreactive T cells and natural killer cells has been detect ed using a novel application of the alamarBlue technique. The assay th at we have developed to detect cell-mediated cytotoxicity is extremely sensitive and specific and requires a significant lower number of eff ector cells than the standard Cr-51 assay. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile condi tions, effector cells may be recovered at the end for further analysis or cell expansion, if desired. Direct comparison of cell-mediated cyt otoxicity measured by the alamarBlue method with the standard Cr-51 re lease assay revealed that the former method is as specific and more se nsitive than the conventional assay. Moreover, very small inter and in tra-assay variations have been observed for alamarBlue cytotoxicity as says, In conclusion, this study shows that the alamarBlue assay is an extremely sensitive, economical, simple and non-toxic procedure to eva luate cell-mediated cytotoxicity that yields accurate results using a limited number of effector cells. Furthermore, since this assay is a o ne-step procedure, and does not involve any risk for the personnel, it may be useful to analyze automatically cell-mediated cytotoxicity in a large number of samples. (C) 1998 Elsevier Science B.V. All rights r eserved.