PMID: 8462666Apr 1, 1993Paper

Spectrophotometric determination of clonogenic capacity of leukemic cells in a semisolid microtiter culture system

Experimental Hematology
C M SchweitzerM M Langenhuijsen

Abstract

In this study we describe a semiautomated clonogenic assay in which human leukemic cell lines (U937 and HL60) are cultured in a semisolid 96-well microtiter culture system and clonogenicity is measured spectrophotometrically in a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT)-assay and sulforhodamine (SRB)-assay. Culture medium with 0.6% (wt/vol) methylcellulose and 10% (vol/vol) fetal calf serum (FCS) appeared to provide optimal culture conditions with a low background absorbance in both colorimetric assays and an optimal linearity between cell number and optical density (OD). An excellent correlation between clonogenic growth of U937 and HL60 cells in the conventional colony-forming unit assay (CFU-assay) and the microtiter CFU-assay was observed. The value of this microtiter CFU-assay was assessed by modulating U937 and HL60 cells with either 1,25(OH)2D3 or cytosine arabinoside (Ara-C). Additionally, the number of living cells was quantitated spectrophotometrically in both MTT- and SRB-assays. Results obtained 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(OH)2D3-modified cells. No s...Continue Reading

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