The effects on cell viability of ursolic acid administration were determined by Sulforhodamine B (SRB) assay or assessed by manual counting with a standard hemocytometer followed by trypan blue staining. T47D, MCF-7 and MDA-MB-231 cells were seeded in a 96-well plate a density of 3,000 cells/well, allowed to adhere overnight, and treated with different doses of ursolic acid (0, 10, 20, 40, 80, 160, and 320 ug/ml) for 3 days at 37°C. Cells were subsequently fixed with 10% trichloroacetic acid for 1 h at 4°C, washed with deionized water and stained with 0.4% SRB dye (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 15 min at room temperature. Cells were then washed with acetic acid and the SRB dye was dissolved with unbuffered Tris at a concentration of 10 mmol/l. Optical density was determined at 540 nm using a plate reader. To create growth curves, breast cancer cells from each cell line were treated with ursolic acid for indicated time (0, 1, 2, 3, 4, 5, and 6 days), and the cell growth was measured after each treatment. The total number of cells was assessed following trypan blue staining at room temperature using a scanning microscope (magnification, ×20; FV1200, Olympus Corp, Japan) and at least 5 areas were randomly selected and analyzed by CellProfiler software (Version 2.1.0; Broad Institute, Cambridge, MA, USA).
Srb dye
Manufactured by Merck Group
Sourced in United States, Italy
SRB dye is a laboratory reagent used for quantitative colorimetric determination of total cellular protein content. It binds to basic amino acid residues in proteins, resulting in a colored complex that can be measured spectrophotometrically.
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Lab products found in correlation
Trichloroacetic acid, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Fv1200, by Olympus (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Ensight plate reader, by PerkinElmer (1 mentions)
Genios plus, by Tecan (1 mentions)
Ensight multimode plate reader, by PerkinElmer (1 mentions)
Victor multiplate reader, by PerkinElmer (1 mentions)
Compusyn software, by ComboSyn (1 mentions)
Trichloroacetic acid, by Carl Roth (1 mentions)
Acetic acid, by Avantor (1 mentions)
Tris base, by Carl Roth (1 mentions)
Genios plus microplate reader, by Tecan (1 mentions)
Trichloroacetic acid tca, by AppliChem (1 mentions)
Trizma base, by Merck Group (1 mentions)
Acetic acid, by Scharlab (1 mentions)
Tris base, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
17 protocols using srb dye
1
Ursolic Acid's Impact on Breast Cancer Cell Viability
2
Cytotoxicity Assessment of Chloroquine
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Huh7 and Mahlavu liver cells were grown in 96-well plates (1000–200 cells/well) in an incubator for 24 h. Both Mahlavu and Huh7 cells were treated with Chloroquine Phosphate (CQ) and water control in 40 μM to 0.3 μM concentrations for 72 h. After fixation with cold 10% (w/v) trichloroacetic acid (MERCK) for an hour at +4°C, plate wells were washed three times with ddH2O. Each well was stained with 50 μl of 0.4%SRB dye(Sigma-Aldrich) and incubated at RT for 10 min. To remove unbound SRB dye, wells were washed with 1% acetic acid for four times and left to air-dying. The protein-bound SRB was solubilized in 100 μl/well 10 mM Tris-base solution, and the absorbance was measured with 96-well plate reader at 515 nm wavelength (ELx800, BioTek).
3
Sulforhodamine B Assay for Cell Viability
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Cell viability was assessed via the SRB assay. Cells were seeded at a density of 6000 cells/well in a 96-well plate. After 48 h of treatment with the indicated treatments, the cells were fixed with 10% trichloroacetic acid (TCA, Sigma, cat. SI-T6399-250G). The fixed cells were washed twice with PBS and stained with sulforhodamine B sodium salt dye (SRB dye, Sigma, cat. S1402) for 30 min. The stained cells were washed three times to remove unbound dye using 1% (v/v) acetic acid. The protein-bound dye was dissolved in a 10 mM Tris-base (Amresco, cat. CPT-0826) solution for absorbance (510 nm) measurement using a multimode microplate reader (Infinite 200 PRO). The relative cell proliferation rate was then compared with the control group.
4
Cell Viability Assay by SRB
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Surviving cell content was measured by SRB assays. Briefly, cells seeded overnight on a 96-well plate were treated for the indicated durations of time, and cell monolayers were fixed with 10% (wt/vol) trichloroacetic acid at 4 °C. After cells were stained with SRB dye (Sigma-Aldrich) for 30 min, excess dye was removed by repeated washing with 1% (v/v) acetic acid. Protein-bound dye was finally dissolved in 10 mM Tris base solution and subject to spectrophotometric measurement of absorbance at 510 nm using a microplate reader.
5
Sulforhodamine B (SRB) Cell Proliferation Assay
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Sulforhodamine B (SRB) assay is a colorimetric test that allows quantifying cellular protein content and it is largely used to indirectly evaluate cell proliferation (Orellana & Kasinski, 2016 (link); Skehan et al., 1990 (link); Vichai & Kirtikara, 2006 (link); Voigt, 2005 (link)). Briefly, cells were seeded and treated as described above in 24‐multiwell plates. At each time point, cells were fixed with 50% trichloroacetic acid (Sigma‐Aldrich, Milan, Italy; T6399) for 2 h at 4°C and washed five times with Milli‐Q water; 0.04% (w/v) SRB dye (Sigma‐Aldrich, Milan, Italy; S1402), dissolved in 1% acetic acid, was added to each well and incubated at room temperature for 30 min; then, each well was washed four times with 1% (v/v) acetic acid and left to air‐dry at room temperature. Finally, 1.2 ml of 10 mM Tris base solution (pH 10.5) was added to each well, and the plate was shaken on an orbital shaker for 10 min to solubilize the protein‐bound dye. The absorbance at 490 nm was detected using a multimode microplate reader (EnSight Plate Reader, PerkinElmer).
6
Sulforhodamine B Assay for Cytotoxicity Quantification
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To quantify cytotoxicity of compounds under investigation, we used the Sulforhodamine B assay.53 (link) Human hepatoma cells (2–4 × 104/well) were cultured in 24-well plates and then treated as described above. At 96 hpi, hepatoma cells were washed with cold PBS and fixed with 10% trichloracetic acid for 30 minutes at 4 °C. After washing the plate with tap water and subsequent drying, tumor cells were stained with SRB dye (Sigma, 0.4 in 1% acetic acid); unbound dye was removed by washing with 1% acetic acid. Protein-bound dye was brought into solution with 10 mmol/l Tris base (pH 10.5), and optical densities were determined using a microtiter plate reader (Tecan Genios Plus; Tecan Deutschland, Crailsheim, Germany) at a wavelength of 550 nm.
7
Quantitative Cell Proliferation Assay
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Sulforhodamine B (SRB) assay is a colorimetric test that allows for quantifying cellular protein content, and it is largely used to indirectly quantify cell proliferation [69 (link)]. Briefly, cells were seeded and treated as described before in 24-multiwell plates. At each time point, cells were fixed with 50% trichloroacetic acid (Sigma-Aldrich, Milan, Italy, cod. T6399) for 2 h at 4 °C, then washed five times with Milli-Q water. Then, 0.04% (w/v) SRB dye (Sigma-Aldrich, Milan, Italy, cod. S1402), dissolved in 1% acetic acid, was added to each well and incubated at room temperature for 30 min; then, each well was washed four times with 1% (v/v) acetic acid and left to air-dry at room temperature. Finally, 1.2 mL of 10 mM Tris base solution (pH 10.5) was added to each well, and the plate was shaken on an orbital shaker for 10 min to solubilize the protein-bound dye. The absorbance at 490 nm was detected using a multimode microplate reader (EnSight Multimode Plate Reader, PerkinElmer, Waltham, MA, USA).
8
Evaluating NQO2-Mediated CB1954 Activation
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HCT116 cells were cultured in McCoy’s 5A (modified) medium with glutamine. Cell culture media was supplemented with 100 units/mL penicillin and 0.1 mg/ml streptomycin. To assess the ability of NQO2 to catalyze redox reactions in cells, activation of CB1954 by NRH was used as a reporter of NQO2 activity (Nolan et al., 2012 (link)). HCT116 or HCT116ΔNQO2 cells were seeded at 1,000 cells per well in 96-well plates and were allowed to attach overnight. Cells were then treated with: 1) 7.8–1,000 μM of CB1954 and 6–200 μM of NRH, 2) 7.8–1,000 μM of CB1954 alone, 3) 4.8–625 μM of NRH alone or 4) nothing (n = 3). Cells were allowed to grow for an additional 48 h before they were counted using sulforhodamine B (SRB) assay, which stains for total protein content of fixed cells (Skehan et al., 1990 (link)). Cells were first fixed with trichloroacetic acid (TCA) for 1 h at 4°C, then washed with water three times. They were then stained with SRB dye (Sigma) for 20 min at room temperature and washed with 1% acetic acid. The dye was re-solubilized in 10 mM Tris base and its absorbance was measured using a multi-reader at 560 nm (Victor multi-plate reader, Perkin Elmer). Absorbance readings of treated cells were normalized against untreated cells, and the data were fitted to a dose-response curve and graphed using Prism (GraphPad Software Inc.).
9
Cytotoxicity Assay with Lanatoside C
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Cells were seeded in 96-well plates (3,500 cells/well) overnight for attachment, treated with the indicated concentrations of lanatoside C for 48 h, then were fixed with 10% TCA (trichloroacetic acid), stained with SRB (sulforhodamine B, 0.4% in 1% acetic acid) for 15 min, and then washed repeatedly with 1% acetic acid. SRB dye was purchased from Sigma Chemical Co. (St Louis, MO, USA). The dye was dissolved with trizma base (10 mM) and then measured by ELISA reader at 510 nm. GI50 was represented the concentration of 50% growth inhibition.
10
Quantitative Cell Viability Assay
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Total protein amount-based Sulforhodamine B (SRB) assays were performed as follows. 5 × 103 (PF338) or 2 × 103 (A375) tumor cells /well were plated on the inner 60 wells of a 96-well plate and first incubated for 48 h. After 72 h of treatment with drugs, 10% TCA was used for fixation, followed by SRB dye (Sigma-Aldrich, St. Louis, MO, United States), and wash out with 1% acetic acid. 10mM Tris puffer dissolved the protein-bound dye and optical density (OD) was read at 570 nm by using a microplate reader (EL800, bioTec Instruments, Winooski, VT, United States). IC50 were calculated by using the CompuSyn software (ComboSyn, Inc., Paramus, NJ). Viability results are illustrated as ratio to control viability. For colony-formation assays, 1,000 tumor cells /well were plated on 6-well plates, incubated for 48 h and subsequently treated every 3–4 days with increasing drug concentrations for 10 days. 10% TCA was used for fixation, followed by SRB dye and wash out with 1% acetic acid. Colonies were counted manually. Experiments were repeated thrice.
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