Fluorescence-imaging assay for cytotoxic plaque formation and for growth toward confluency of adherent cells.
A nondestructive fluorescence-imaging assay is described for quantitating the number and size of plaques formed over time by cytotoxic effector cells in a monolayer of target cells. It can also be used to assay the growth of adherent cells toward confluence. The method involves the use of fluorescein conjugated to high molecular weight dextran. The dextran is excluded by adherent cells, thereby making the medium around cells more fluorescent than the cells themselves. The area of the plate that is fluorescent can be determined by confocal fluorescence imaging microscopy. With this new method, changes in the confluency of adherent cells or in the number and area of cytotoxic plaques can be assayed repeatedly over an extended period of time, without manipulation of the cells or of the medium.
Gericke
G
G
Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Benoit
N
N
Orndorff
K A
KA
Givan
A L
AL
Ericson
S G
SG
D002448:Cell Adhesion; D003602:Cytotoxicity, Immunologic; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
D016650:Fluorescein-5-isothiocyanate