A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry

Jodi E. Goldberg, Steven W. Sherwood, Carol Clayberger*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

75 Scopus citations

Abstract

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin V(neg) to annexin V(hi) is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalJournal of Immunological Methods
Volume224
Issue number1-2
DOIs
StatePublished - Apr 22 1999

Keywords

  • Annexin V
  • Cr release
  • Flow cytometry
  • Human cytotoxic T cells

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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