Srb staining solution

Sulforhodamine B (SRB) assay was used to assess the cytotoxicity of the compounds [17 (link)]. Briefly, HT-29 and CCD-18Co cells were seeded in 96-well-plate at 1.5 × 10⁵ cells/ml respectively and were incubated at 37 °C in 5% CO₂ for 24 h. 100 µl of six concentrations of serially diluted compounds (0.94–30 µg/ml) were added in their respective wells. Hydrogen peroxide (10 mM) was used as the positive control. The plate was incubated for 48 h. Cells were fixed with 50 µl of 50% cold (4 °C) trichloroacetic Acid (TCA) and incubated at 4 °C for 1 h. The plates were washed five times with tap water and air-dried before staining with 100 µL of 0.4% (w/v) SRB staining solution (Sigma-Aldrich, St. Louis, MO, USA). Further incubation was done for 30 min at room temperature. Subsequently, the plates were washed three times with 1% (v/v) acetic acid to remove any unbound stains. After air-dried, 200 µL of 10 mM trizma base were added into each well and agitated for 15 min. Absorbance was read with a microplate reader with Microplate Manager® Software at the wavelength of 564 nm. All experiments were carried out in triplicates.

Cell viability was quantified by sulforhodamine B (SRB) staining, which was used to determine the total protein content. In detail, SCP-1 cells were fixed with ice-cold 99% ethanol (–20 °C) for 1 h. Subsequently, cells were washed 3 times with PBS and stained with SRB staining solution (0.4% SRB in 1% acetic acid, Sigma-Aldrich, Munich, Germany) for 30 min at room temperature. Then, cells were washed 4 to 5 times with 1% acetic acid to remove unbound SRB. For quantification, the bound SRB was resolved with 10 mM unbuffered TRIS solution (pH~10.5). The OD was measured with a microplate reader at 565–690 nm [4 (link)].