A new microcellular cytotoxicity test based on calcein AM release

Hum Immunol. 1993 Aug;37(4):264-70. doi: 10.1016/0198-8859(93)90510-8.


We present a microtest for cell-mediated immunity, based on the use of the Tarasaki tray and calcein AM vital dye. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read at the rate of 1 second per test using a fluorimeter attached to a microscope. Each reaction was also confirmed visually with the use of ethidium bromide as a counterstain for dead cells. The calcein AM dye used to stain the living cells was shown to have a low spontaneous leakage rate--less than 15% in 4 hours at 37 degrees C. Dilutions of targets stained by calcein AM had a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs were readily detectable by this microtest. Quantitation of killing and kinetic analysis was readily performed with this test system. A significant positive correlation to 51Cr-release results was found. We conclude that the microtest should find wide application in studies of cell-mediated immunity.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Cytotoxicity Tests, Immunologic / methods*
  • Cytotoxicity, Immunologic
  • Fluoresceins
  • Humans
  • Immunity, Cellular
  • Indicators and Reagents
  • Killer Cells, Lymphokine-Activated / immunology
  • Killer Cells, Natural / immunology
  • Reproducibility of Results
  • T-Lymphocytes, Cytotoxic / immunology
  • Tumor Cells, Cultured


  • Fluoresceins
  • Indicators and Reagents
  • carboxyfluoresceindiacetate
  • fluorexon