Improved bioassay for the detection of transforming growth factor-beta 1 and beta 2 in malignant gliomas

Growth inhibition assays using radioisotope or dye are used to detect transforming growth factor-beta (TGF-beta). Here, we describe a modified bioassay using crystal violet for the quantitative detection of TGF-beta 1 and TGF-beta 2. 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-beta 1 and TGF-beta 2 increased approximately twofold by using only 500 Mv1Lu cells in microtiter wells. The bioassay was used to measure TGF-beta activity in the culture supernatant from glioblastoma cells. Culture supernatants were untreated or acid-activated to quantify the active or total TGF-beta, and neutralized with anti-TGF-beta 1 and/or anti-TGF-beta 2 antibody to measure the activity. Both TGF-beta 1 and TGF-beta 2 were detected in the untreated and acid-activated supernatants, and the amounts were calculated by extrapolating from the known recombinant TGF-beta 1 or TGF-beta 2 dilution curve. Our results show that the modified bioassay using crystal violet can measure the levels of TGF-beta 1 and TGF-beta 2 in culture supernatants from malignant glioma cells.