Sulforhodamine b srb solution

Mahlavu,
FOCUS, SNU475 (1000 cell/well in 150 μL/well); Huh7, MCF7 (2500
cell/well in 150 μL/well); HepG2, Hep3B, SKBR3 (3000 cell/well
in 150 μL/well); PLC-PRF-5, Hep40 (5000 cell/well in 150 μL/well);
MDA-MB-231, MDA-MB-468, MCF10A (6000 cell/well in 150 μL/well);
and ZR-75 (7500 cell/well in 150 μL/well) were cultured in 96-well
plates and were inoculated in an incubator for 24 hours. The compounds
were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO)
as a 20 mM stock solution. The treatment of cells with the compounds
was done in a concentration range starting from 40 to 2.5 μM.
The compounds which were below 2.5 μM were tested in a concentration
range from 2.5 to 0.15 μM. End of the 72h compound treatment,
the cells were fixed using 10% (v/v) trichloroacetic acid (MERCK)
for an hour and washed with ddH₂O, and left for air-drying.
The fixed plates were stained with 50 μL of sulforhodamine B
(SRB) solution (Sigma) at RT for 10 min. To remove unbound SRB dye,
acetic acid was used to wash cells three times and left for drying.
The protein-bound SRB was solubilized with 10 mM Tris-base (Sigma)
and their absorbance was measured with a 96-well plate reader at 515
nm wavelength (ELx800, Biotek). The IC₅₀ values of compounds
were calculated in comparison with control group DMSO. Data with R² values larger than 0.9 are considered significant.

Huh7, MCF7 (2500 cell/well in 150 µl/well) and Mahlavu (1000 cell/well in 150 µl/well) cells were plated in 96-well plates and were grown in incubator for 24 hours. The compounds were dissolved in DMSO (Sigma, St Louis, MO, USA) as 20 mM stock solution. The compounds which were below 2.5 µM were tested in a concentration range of starting from 2.5 µM to 0.015 µM. Cells were fixed using 10% (v/v) trichloroacetic acid (Sigma ) for an hour after the end of 72 h incubation time. The fixed plates were dried and fixed cells were stained with sulforhodamine B (SRB) solution (Sigma) (50 µl of a 0.4% (m/v) of SRB in 1% acetic acid solution (Sigma)) for 10 min. In order to remove unbound SRB dye, cells were washed with 1% acetic acid three times and left for air-drying. The protein bound SRB dye was dissolved in 10 mM Tris-base (Sigma) and absorbance was measured with 96-well plate reader at 515 nm. The IC₅₀ values were calculated and the cells treated with DMSO alone were used as control. All experiments were done in triplicate. Data with R² values >0.9 was considered significant.