Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which < 200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37 degrees with 5% CO2 at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appreciably after 2--3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.