ASSAY OF ANTICANCER DRUGS IN TISSUE-CULTURE - COMPARISON OF A TETRAZOLIUM-BASED ASSAY AND A PROTEIN-BINDING DYE ASSAY IN SHORT-TERM CULTURES DERIVED FROM HUMAN-MALIGNANT GLIOMA

Because of the methodological difficulties associated with the MTT ass ay in screening short-term cultures derived from human malignant gliom a, a chemosensitivity assay based on the protein staining using sulfor hodamine B (SRB) has been optimized for use with these cells. SRB at a fixed dye concentration achieved maximal staining density at 20 min f or most cell lines and this intensity was not further increased by usi ng dye concentrations above 0.2%. A delay in staining after fixation d id not significantly decrease staining intensity, but delay in dye ext raction after fixation and staining did, There was an excellent quanti tative and qualitative linear relationship between cell number determi ned by either the SRB essay or by cell counting, but not with the MTT assay which consistently underestimated the number of cells in assay p lates. The MTT assay appeared to be incapable of detecting less than a bout 150 cells/well, while these small numbers of cell were readily de tectable by either cell counting or SRB staining, There was a close co rrelation between chemosensitivity values derived from the MTT and SRB assays for procarbazine, CCNU and vincristine when the endpoint is ta ken as either the ID25, ID50 or ID75. The results indicate that the SR B is capable of producing broadly similar results to the MTT assay, bu t is more sensitive in the detection of small numbers of cells with a linear relationship between cell number and SRB staining intensity ove r a wide range of cell numbers, It is capable of producing data from s hort-term cultures from malignant glioma and offers technical advantag es over the MTT assay in that plates may safely be stored at certain p oints during the assay without the need for immediate processing. The SRB assay provides a useful alternative to the MTT assay for determini ng the sensitivity of short-term cultures of human glioma to cytotoxic drugs.