Sulforhodamine B (SRB) Assay

To ensure that effects observed in ALP assays were not caused by cytotoxicity, SRB cell viability assays were carried out in parallel. As with estrogenicity assays, 10,000 cells/well were seeded in 96-well plates, allowed to grow for 48 h and incubated with test solutions for another 48 h. Afterwards, cells were fixed by addition of 50% (v/v) trichloroacetic acid and 1 h incubation at 4°C. After 2 times washing with tab water and 2 times of washing with 1% (v/v) acetic acid, plates were dried overnight, incubated with SRB reagent for 45 min, washed twice with water, twice with 1% acetic acid and again dried overnight. Lastly, 10 mM TRIS base buffer was added to dissolve protein-bound SRB and the absorbance of each well was measured at 570 nm with a PerkinElmer Victor 3V^TM plate reader.