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Sulforhodamine b srb solution

Manufactured by Merck Group

Sulforhodamine B (SRB) solution is a laboratory reagent used in colorimetric assays to measure cell viability and proliferation. It is a bright pink dye that binds to basic amino acid residues in cellular proteins, allowing the quantification of cellular biomass. The SRB solution is commonly used in various cell-based assays, such as cytotoxicity, drug screening, and cell growth studies.

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4 protocols using sulforhodamine b srb solution

1

Cytotoxicity Screening of Compounds

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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.
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2

Cytotoxicity Evaluation of Compounds

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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.
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3

Quantifying Cell Proliferation via SRB Assay

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Cells were transfected with siRNAs using Lipofectamine RNAiMax reagent (Invitrogen) according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were seeded into 5 24-well plates. One, 2, 3, 4 and 5 days after seeding, 4 wells of cells for each data point (e.g., siRNA or control) were fixed in 10% TCA (Sigma-Aldrich) culture, stained with 0.4% sulforhodamine-B solution (SRB) (Sigma-Aldrich), and washed with 1% acetic acid. Absorbance was recorded with a spectrometer at 490 nm.
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4

Cytotoxic effects of MEDI5117 on HNSCC

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For these studies, we plated 2 × 103 HNSCC cells per well (UM-SCC-11B or UM-SCC-22B, kindly provided by Dr. T. Carey) and exposed them to 0.01 to 100 μg/ml MEDI5117 or 100 μg/ml control IgG (R347). After 24 to 72 hours, cells were fixed with 50% trichloroacetic acid and stained with 0.4% sulforhodamine-B solution (SRB; Sigma Aldrich). Unbound SRB dye was removed by washing with 1% acetic acid. Plates were air-dried and bound SRB was resolubilized in 10 mM unbuffered Trizma base. Absorbance was analyzed on a microplate reader at 560 nm (Genios Tecan). Test results were normalized against initial plating density and IgG controls. Quadruplicate wells per condition were evaluated and are representative of at least two independent experiments.
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