Sulforhodamine b srb dye

Skin-derived fibroblasts of patient #1 and of a non-carrier sibling (provided by Dr. Reza Maroofian) and of patient #2 were grown in high-glucose DMEM supplemented with 10% fetal bovine serum, 2mM glutamine, 5mM non-essential amino acids and 1% (v/v) MEM vitamins (ThermoFisherScientific). Growth measurements were performed in 96-well plates and quantified by staining with the sulforhodamine B (SRB) dye (Sigma). Cells were fixed with 10% ice-cold trichloroacetic acid for 1 hour at 4°C, washed 4 times with water, and air-dried overnight at room temperature. Protein content was stained with 50μl of 0.04% SRB made in 1% acetic acid for 1 hour. Excess dye was removed by four washes with 1% acetic acid, and wells were air-dried for ≥2 hours. Stained proteins were suspended in 50μl of 10mM Tris base for 10 min. Absorbance was measured at 560nm with a microplate reader.

Sulforhodamine B (SRB) dye (Sigma-Aldrich, Chemie GmbH, Munich, Germany) was used to test the effects of selective inhibitors on cell growth and viability of SP cells. The regorafenib were dissolved in dimethyl sulfoxide (DMSO) before diluting with growth medium to a final DMSO concentration of 0.05%. The HCT-116R and DLD-1R cells were seeded into 96 well plates in growth medium at 3000 cells/well. After 24 h the medium was replaced with fresh growth medium containing the regorafenib. The cells were incubated for another 48 h. The cells were fixed with trichloroacetic acid (TCA) by gently adding 50 μL TCA (50%) to each well to a final TCA concentration of 10% with subsequent incubation for 1 h at 4 °C. The plates were then washed 5 times with tap water and air dried. The dried plates were stained with 100 μL of 0.4% (w/v) SRB prepared in 1% (v/v) acetic acid for 10 min at room temperature. The plates were rinsed quickly 4 times with 1% acetic acid to remove unbound dye, and then air dried until no moisture was visible. The bound dye was solubilized in 20 mM Tris-base (100 μL/well) for 5 min on a shaker. Optical densities were read on a microplate reader (Molecular Devices, Sunnyvale, CA) at 562 nm.