SPECTROPHOTOMETRIC DETERMINATION OF CLONOGENIC CAPACITY OF LEUKEMIC-CELLS IN A SEMISOLID MICROTITER CULTURE SYSTEM

In this study we describe a semiautomated clonogenic assay in which hu man leukemic cell lines (U937 and HL60) are cultured in a semisolid 96 -well microtiter culture system and clonogenicity is measured spectrop hotometrically in a 3-[4,5-dimethylthiazol-2-yI]-2, 5 diphenyl tetrazo lium bromide (MTT)-assay and sulforhodamine (SRB)-assay. Culture mediu m with 0.6% (wt/vol) methylcellulose and 10% (vol/vol) fetal calf seru m (FCS) appeared to provide optimal culture conditions with a low back ground absorbance in both colorimetric assays and an optimal linearity between cell number and optical density (OD). An excellent correlatio n between clonogenic growth of U937 and HL60 cells in the conventional colony forming unit assay (CFU-assay) and the microtiter CFU-assay wa s observed. The value of this microtiter CFU-assay was assessed by mod ulating U937 and HL60 cells with either 1,25(OH)(2)D-3 or cytosine ara binoside (Ara-C). Additionally, the number of living cells was quantit ated spectrophotometrically in both MTT- and SRB-assays. Results obtai ned by either method did not differ significantly (p<0.001). In liquid culture, however, significantly (p<0.001) less reduction of cellular growth was observed with 1,25(0H)(2)D-3-modified cells. No significant differences between the liquid and semisolid assay systems could be o bserved in the presence of Ara-C. In conclusion, the microtiter clonog enic assay provides a rapid and objective way of measuring clonogenic capacity of (leukemic) cell lines. The semiautomated clonogenic assay will be useful to assess large series of immune modulations and/or cyt ostatic drug screening.