title: A new microcellular cytotoxicity test based on calcein AM release.
abstract: 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.
authors.affiliation: UCLA Tissue Typing Laboratory, Department of Surgery, UCLA School of Medicine 90024.
authors.forename: X M
authors.initials: XM
authors.lastname: Wang
authors.forename: P I
authors.initials: PI
authors.lastname: Terasaki
authors.forename: G W
authors.initials: GW
authors.lastname: Rankin
authors.forename: D
authors.initials: D
authors.lastname: Chia
authors.forename: H P
authors.initials: HP
authors.lastname: Zhong
authors.forename: S
authors.initials: S
authors.lastname: Hardy
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D005452:Fluoresceins; D006801:Humans; D007111:Immunity, Cellular; D007202:Indicators and Reagents; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology; D015203:Reproducibility of Results; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D005452:Fluoresceins; D007202:Indicators and Reagents; C027780:carboxyfluoresceindiacetate; C007740:fluorexon