An in Vitro Assay to Measure the Viability of KHT Tumor Cells Not Previously Exposed to Culture Conditions

An in vitro colony assay which measures the viability of KHT fibrosarcoma cells is described. Single cell suspensions are prepared from tumors growing in C3H mice with no previous adaptation for in vitro growth. The assay involves incubating cells at 37° for 2 wk in Petri dishes containing a semisolid matrix of α medium, fetal calf serum, and Bacto agar. Colonies containing 20 or more cells are then counted under a microscope. The plating efficiency of the assay ranges from 5-40% based on the number of viable cells plated as determined by dye exclusion. However, this dye-exclusion technique may overestimate the number of viable cells since the plating efficiency of cells separated by velocity sedimentation ranges from 50-90%. Survival curves have been determined for cell suspensions irradiated in vitro in air or nitrogen.${\rm D}_{0}\text{'}{\rm s}$ of 167 and 478 rads and extrapolation numbers of 1.6 and 1.4 were obtained for aerobic and anoxic curves, respectively. These numbers are in reasonable agreement with previous values obtained for this tumor by others using in vivo assays. Survival curves for tumors irradiated in vivo and assayed in vitro or in vivo are also in good agreement.