A novel one-step, highly sensitive fluorometric assay to evaluate cell- mediated cytotoxicity

Marcelo M. Nociari, Assaf Shalev, Petros Benias, Carlo Russo

Research output: Contribution to journalArticlepeer-review

300 Scopus citations

Abstract

In this study, a fluorometric method using alamarBlue has been developed 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 cells is quantified by comparing the total number of viable cells in wells containing effector and targets together, with wells where target and effector cells were separately seeded. Cell-mediated cytotoxic activity by alloreactive T cells and natural killer cells has been detected using a novel application of the alamarBlue technique. The assay that we have developed to detect cell- mediated cytotoxicity is extremely sensitive and specific and requires a significant lower number of effector cells than the standard 51Cr assay. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile conditions, effector cells may be recovered at the end for further analysis or cell expansion, if desired. Direct comparison of cell- mediated cytotoxicity measured by the alamarBlue method with then standard 51Cr release assay revealed that the former method is as specific and more sensitive than the conventional assay. Moreover, very small inter and intra- assay variations have been observed for alamarBlue cytotoxicity assays. In conclusion, this study shows that the alamarBlue assay is an extremely sensitive, economical, simple and non-toxic procedure to evaluate cell- mediated cytotoxicity that yields accurate results using a limited number of effector cells. Furthermore, since this assay is a one-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.

Original languageEnglish
Pages (from-to)157-167
Number of pages11
JournalJournal of Immunological Methods
Volume213
Issue number2
DOIs
StatePublished - Jun 1998
Externally publishedYes

Keywords

  • AlamarBlue
  • CTL
  • Cell-mediated cytotoxicity
  • Fluorescence
  • NK
  • Natural killer activity

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