The cells (MDA-MB-231, NCCS, Pune) were grown in appropriate medium containing 10 % fetal bovine serum (Cat#092916854, M.P. Biochemicals) and 2 mM l -glutamine (Cat #12800-017, GIBCO) These were incubated at 37 °C, 5 % CO2, with 5000 cells in each well of the 96 well plate. The test drug was diluted and different concentrations were prepared with further incubation for 48 h. The addition of TCA (Cat #2844 M, Fisher Scientific) in 50 μl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) for assay termination. The well washed and air dried sample is further treated with the Sulforhodamine B (SRB) (Cat# S9012 CAS- 3620, SIGMA) solution (50 μl) at 0.4 % (w/v) in 1 % acetic acid kept for 20 min at room temperature. Unbound dye recovered and residual dye washed off and treated with trizma base (Cat# T1378, SIGMA) before OD measured at 540 nm (Model# ELx808, BIOTEK instruments Inc). Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells × 100 [27 (link)].
Sulforhodamine b srb
Manufactured by Merck Group
Sourced in United States, Germany, Italy, China, United Kingdom, Macao
Sulforhodamine B (SRB) is a bright pink, water-soluble, xanthene dye that is commonly used in biological research applications. It is a sensitive and reliable stain for quantifying cellular protein content in cell proliferation and cytotoxicity assays.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Trichloroacetic acid tca, by Merck Group (21 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (20 mentions)
Acetic acid, by Merck Group (18 mentions)
Trypan blue, by Merck Group (11 mentions)
Tris base, by Merck Group (10 mentions)
Ellipticine, by Merck Group (9 mentions)
Trichloroacetic acid, by Merck Group (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Trizma base, by Merck Group (7 mentions)
Crystal violet, by Merck Group (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Doxorubicin, by Merck Group (5 mentions)
Paclitaxel, by Merck Group (5 mentions)
Penicillin, by Merck Group (5 mentions)
Streptomycin, by Merck Group (5 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (5 mentions)
178 protocols using sulforhodamine b srb
1
SRB Cytotoxicity Assay for MDA-MB-231 Cells
2
Osteoblast Viability Assay: Imatinib vs. Nilotinib
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In the in vitro system, the following three sample groups were examined: Imatinib-treated, nilotinib-treated and untreated (control) osteoblast cell cultures.
The adequate incubation time and drug concentration were defined using a cell viability assay. Different imatinib (Glivec/Gleevec, STI571, CGP 57148B; Novartis, Basel Switzerland) and nilotinib (Tasigna; Novartis) concentrations (30 nM-20 µM) were administered to 40% confluent MC3T3-E1 cells for various incubation times (1–6 days) in 96-well plates. After removal of culture medium, cells were fixed with 100 µl/well trichloroacetic acid (Sigma-Aldrich) for 30 min. Then, cells were stained with 0.4% sulforhodamine-B (SRB, Sigma-Aldrich) protein dye solution for 30 min. After the excess dye solution removal, cell culture plates were rinsed with 1% acetic acid solution four times and dried at room temperature. The bound SRB was dissolved in 100 µl of 10 mM Trisma-Sol (Sigma-Aldrich) and the cell culture plates were shaken for 5 min. The measurements were performed by Multiskan Spectrum V1.2 1500-636 device (Thermo Fisher Scientific Inc, Waltham MA USA) at 520 nm. Three parallel measurements on 24-well cell culture plates were performed twice in all experiments.
The adequate incubation time and drug concentration were defined using a cell viability assay. Different imatinib (Glivec/Gleevec, STI571, CGP 57148B; Novartis, Basel Switzerland) and nilotinib (Tasigna; Novartis) concentrations (30 nM-20 µM) were administered to 40% confluent MC3T3-E1 cells for various incubation times (1–6 days) in 96-well plates. After removal of culture medium, cells were fixed with 100 µl/well trichloroacetic acid (Sigma-Aldrich) for 30 min. Then, cells were stained with 0.4% sulforhodamine-B (SRB, Sigma-Aldrich) protein dye solution for 30 min. After the excess dye solution removal, cell culture plates were rinsed with 1% acetic acid solution four times and dried at room temperature. The bound SRB was dissolved in 100 µl of 10 mM Trisma-Sol (Sigma-Aldrich) and the cell culture plates were shaken for 5 min. The measurements were performed by Multiskan Spectrum V1.2 1500-636 device (Thermo Fisher Scientific Inc, Waltham MA USA) at 520 nm. Three parallel measurements on 24-well cell culture plates were performed twice in all experiments.
3
Cytotoxicity Assay of AXL Inhibitor R428
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3 × 103 cells per well were seeded into 96-well plates. The next day, cells were treated with serially diluted (final concentration: from 10μM to 0.312μM) AXL inhibitor R428 (HY-15150, MedChemExpress, Princeton, NJ, USA) dissolved in DMSO. After 72 h of incubation, cells were fixed with 10% ice-cold trichloroacetic acid (TCA) (T6399, Sigma-Aldrich, St. Louis, MO, USA) and incubated at −20 °C for 20 min. Fixed cells were stained with 0.4% Sulforhodamine B (SRB) (S1402, Sigma-Aldrich, St. Louis, MO, USA) in 1% acetic acid (CAS:64-19-7, Sigma-Aldrich, St. Louis, MO, USA ) solution for 20 min. After staining, excess SRB was removed with 1% acetic acid, and the plates were left to dry. The absorbance in each well was measured after adding 50 μL of 10 mM Tris-Base (T1503, Sigma-Aldrich, St. Louis, MO, USA). The OD was measured at 512 nm using Varioskan, FLASH (Thermo Fisher Scientific, Waltham, MA, USA).
4
PARP Inhibitor BMN673 Cytotoxicity Assay
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The PARP inhibitor BMN673 was a gift from Biomarin Pharmaceuticals, USA. Survival assays were carried out as previously described [33 (link), 34 (link)]. In brief, the previously transfected cells were seeded in six-well plates in triplicates at a concentration of 1000 cells per well. The PARP inhibitor treatment was started after 24 h and cells were continuously exposed to the PARP inhibitor (10–11 to 10–6 M) and the medium was replaced every 3 to 5 days. The controls were treated with the vehicle substance (DMSO). Cells cultures were grown until the controls reached 80–90% confluence after 10 to 15 days and then fixated with TCA 10%. After fixation, cell cultures were stained with sulforhodamine B (SRB) (Sigma) and a colorimetric assay was performed as described previously [33 (link)].
5
siRNA Knockdown of ZNF703 Impacts Cell Proliferation
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Cell lines were transfected with short-interfering RNA (siRNAs, 30 nM final concentration) in 6-well plates with RNAiMAX (Invitrogen) according to the manufacturer’s instructions and harvested 48 hours after transfection, which could be cultured to enter following experiments. Target sequences for the siRNA of ZNF703: sense strand-5’ CCACACACUUUGGGCCUAA dTdT 3’; antisense-strand-3’ dTdT GGUGUGUGAAACCCGGAUU 5’. Non-targeting control siRNA was designed and synthesized by Guangzhou RuiBoBio (Guangzhou, China). Proliferation assay and colony-forming assay were performed as previously described [22 (link)]. Cell proliferation was measured by sulforhodamine B (SRB) (Sigma) assay. Relative growth was calculated as the value relative to controlled cells. In colony-forming assay, cells were seeded into 6-well plates (1000 cells per well). After several proper days, colonies were fixed in 10% acetic acid, 10% methanol and 80% ddH2O, and then stained with crystal violet (0.5% w/v).
6
Antioxidant and Cytotoxic Evaluation
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Folin-Ciocalteu reagent, gallic acid, quercetin, ascorbic acid, curcumin, β-sitosterol, lupeol, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3-(4, 5-dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sulforhodamine B (SRB), Hoechst 33258 dye, crystal violet, propidium iodide were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade and purchased from the usual sources.
7
Cell Survival and Proliferation Assay with Drug and Radiation
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To evaluate the drug’s effects on cell survival and proliferation, cells were seeded in 96-well plates. After attachment overnight on 37 °C, cells were treated with drug concentrations of 0; 0.5; 1; 2.5; 5 µM of BKM120 and 0; 0.1; 0.25; 0.5; 1; 2.5; 5; 10 µM of GDC0980. Two hours after drug administration plates were irradiated with doses of 0, 2, 4 or 6 Gy using a Baltograph (199 kV, 15 mA, Balteau NDT, Belgium). The drug was replaced by fresh full medium after 72 h (h) for BKM120 and 24 h for GDC0980. The time of exposure has been based on pilot studies32 (link),68 (link),73 (link),74 (link). One week after seeding, cells were fixed and stained with sulforhodamine B (SRB, Sigma-Aldrich). The incorporated SRB was liberated from cells in a tris-base solution, and optical densities were determined at 570 nm. Cell survival was determined as the relative absorption of SRB in treated wells in comparison to controls.
8
In Vitro Evaluation of Famitinib Malate
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Famitinib Malate was provided by Jiangsu Hengrui Medicine Co., Ltd. HS‐10296 was obtained from Jiangsu Hansoh Pharmaceutical Co., Ltd. Sulforhodamine B (SRB) was purchased from Sigma‐Aldrich. Cell counting kit‐8 (CCK‐8) was purchased from Dalian Meilun Biotechnology Co., Ltd.
Antibodies against EGFR, phospho‐EGFR(Tyr1173), AKT, phospho‐AKT (Ser473), ERK1/2, phosphor‐ERK1/2 (Thr202/Tyr204), PARP, caspase‐3 and GAPDH were purchased from Cell Signaling Technology. Antibody against ERK1/2 was purchased from Santa Cruz Biotechnology. The anti‐CD31 antibody was purchased from Abcam.
Antibodies against EGFR, phospho‐EGFR(Tyr1173), AKT, phospho‐AKT (Ser473), ERK1/2, phosphor‐ERK1/2 (Thr202/Tyr204), PARP, caspase‐3 and GAPDH were purchased from Cell Signaling Technology. Antibody against ERK1/2 was purchased from Santa Cruz Biotechnology. The anti‐CD31 antibody was purchased from Abcam.
9
Phytochemical Evaluation and Bioactivity Assessment
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Acetonitrile (HPLC grade), 95% ethanol, 37% hydrochloric acid, sodium hydroxide, sulfuric acid, and dichloromethane were purchased from RCI Labscan, Bangkok, Thailand. Gallic acid, Folin–Ciocalteau phenol reagent, aluminum chloride, sodium acetate trihydrate, ferric chloride solution, ferrous sulfate heptahydrate, and potassium persulfate sodium carbonate were obtained from Loba Chemie, Mumbai, India. A549 (ATCC® CCL-185TM) human lung adenocarcinoma cell line was purchased from the American Type Culture Collections (ATCC; Manassas, VA, USA). HaCaT cell line was purchased from Pacific Science, Co., Ltd. (Bangkok, Thailand). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, 100 U/mL and 100 µg/mL streptomycin, and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA). Dragendorff’s reagent, phosphotungstic acid, Gallic acid, DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine, ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and sulforhodamine B (SRB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC and PI solution were purchased from BD Biosciences, CA, USA). DCF-DA was purchased from Sigma Merck KGaA (Darmstadt, Germany).
10
Thiazolidinone Derivatives Screening
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The thiazolidinone derivatives used for compound combination screening in this study were reported previously.13 (link) All compounds were dissolved in DMSO and stored at 4 °C as stocks. Each compound was diluted before use. Compound combinations were prepared by mixing the four compounds with equal molar ratio. RPMI 1640, fetal bovine serum, penicillin, streptomycin, and all other tissue culture reagents were obtained from Life Technologies (Grand island, NY, USA). The Guava Nexin Assay reagent and Guava EasyCyte MitoPotential kit were obtained from Millipore (Billerica, MA, USA). Caspase-Glo 8 assay kit, Caspase-Glo 9 assay kit and Apo-ONE Homogeneous Caspase-3/7 Assay Kit were from Promega (Promega Biotech CO., Ltd, Madison, WI, USA). The Mitochondria isolation kit was obtained from Pierce Biotechnology (Rockford, IL, USA); Sulforhodamine B (SRB) and other chemicals reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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