A NEW FLUOROMETRIC ASSAY FOR DETERMINATION OF OSTEOBLASTIC PROLIFERATION - EFFECTS OF GLUCOCORTICOIDS AND INSULIN-LIKE GROWTH-FACTOR-I

A novel fluorometric proliferation assay, AlamarBlue (AB), was used to study the proliferative capacity of isolated human osteoblasts (hOBs) . AB is an oxidation-reduction indicator that yields a fluorescent sig nal in response to metabolic activity. The assay was performed by repl acing the experiment media in a microtiter plate with a 10% AB solutio n and measuring fluorescence after a 3-8-hour incubation. The assay wa s optimized with respect to incubation time, cell density, and AB conc entration. When the results of the AB assay were compared with cell co unting in a Burker chamber there were consistently goad correlations ( r > 0.9), regardless of the agonist with which the cells were treated. The mean intraassay coefficient of variance (CV) values were 9.9-11.8 % in experiments where osteoblasts were treated for 12 days with insul in-like growth factor-I (IGF-I; 100 nM), or dexamethasone (1 mu M). IG F-I dose dependently, at and above I nM, stimulated proliferation of h OBs. This effect was detectable after 3 days and reached 130-140% of u ntreated controls after 12 daps in culture. The effects of dexamethaso ne (DEX) on the proliferation rate of hOBs were more complex. In short -term cultures, 3 days, DEX dose dependently stimulated proliferation. However, at and above 6 days, DEX exerted a biphasic effect, with sti mulation seen at 1-10 nM and a marked inhibition of cell proliferation at and above 100 nM. dexamethasone, hydrocortisone, prednisolone, and deflazacort had almost identical biphasic effects on osteoblastic pro liferation in 12 day cultures with a stimulation seen at 1-10 nM, and a marked inhibition down to 50-60% of untreated controls at and above 100 nM. When IGF-I (0.1-100 nM; 12 day culture) was combined with diff erent doses of DEX, IGF-I still dose dependently stimulated the prolif eration rate in hOBs regardless of the amount of DEX added. The stimul atory effect of DEX (10 nM, 12 days culture) was additive to the effec t of 100 nM IGF-I. We conclude that AB is an easy and reliable assay f or osteoblastic cell proliferation well suited for large scale studies of cell growth using small amounts of cells, and that IGF-I partly re verses the glucocorticoid-induced inhibition of osteoblastic prolifera tion.