抄録
Growth inhibition assays using radioisotope or dye are used to detect transforming growth factor-β(TGF-β). Here, we describe a modified bioassay using crystal violet for the quantitative detection of TGF-β1 and TGF-β2. The procedure is based on staining Mv1Lu mink lung epithelial cells with crystal violet, followed by measurement of the absorbance at 570 nm in individual wells of a 96-well microtiter plate. The number of Mv1Lu cells correlated with the eluted dye intensity. The sensitivity of the bioassay to recombinant TGF-β1 and TGF-β2 increased approximately twofold by using only 500 Mv1Lu cells in microtiter wells. The bioassay was used to measure TGF-β3 activity in the culture supernatant from glioblastoma cells. Culture supernatants were untreated or acid-activated to quantify the active or total TGF-β, and neutralized with anti-TGF-β1 and⁄or anti-TGF-β2 antibody to measure the activity. Both TGF-β1 and TGF-β2 were detected in the untreated and acid-activated supernatants, and the amounts were calculated by extrapolating from the known recombinant TGF-β1 or TGF-β2 dilution curve. Our results show that the modified bioassay using crystal violet can measure the levels of TGF-β1 and TGF-β2 in culture supernatants from malignant glioma cells.
