A NEW MICROCELLULAR CYTOTOXICITY TEST BASED ON CALCEIN AM RELEASE

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 cell s needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read a t the rate of 1 second per test using a fluorimeter attached to a micr oscope. Each reaction was also confirmed visually with the use of ethi dium 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 s tained by calcein AM had a linear relationship with measured fluoresce nce values. NK cells, LAKs, and CTLs were readily detectable by this m icrotest. Quantitation of killing and kinetic analysis was readily per formed with this test system. A significant positive correlation to Cr -51-release results was found. We conclude that the microtest should f ind wide application in studies of cell-mediated immunity.