Cell viability was assessed by MTS proliferation assay kit (Promega), according to that manufacturer’s protocol. Briefly, 1–4 × 104 cells were plated in a 96-well plate in 100 μL growth medium to make the cell population at ∼80% confluence. After 24 h, 1 μL drug solution was added to each well. DMSO was used as a control compound. After another 48- to 72-h incubation (37°C, 5% CO2), 20 μL of freshly prepared 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/phenazine methosulfate (PMS) solution was added to each well, and the plate was returned to the incubator for 1 h. Absorbance at 490 nm was recorded, and the result was analyzed by GraphPad Prism 5. GI50 values represent mean ± SEM for at least two separate experiments performed in triplicate. Culture conditions and experimental protocols to determine GI50 values for compound 1 in the primary murine Ptch-CKO cells were as previously described.28 (link), 29 (link), 30 (link)
Mts proliferation assay kit
Manufactured by Promega
Sourced in United States
The MTS proliferation assay kit is a colorimetric method used to measure cell proliferation and cell viability in vitro. The assay involves the bioreduction of a tetrazolium compound, which produces a colored formazan product that can be quantified using a spectrophotometer.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Prism 5, by GraphPad (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Drr036a, by Takara Bio (1 mentions)
Protein extraction buffer, by Beyotime (1 mentions)
Recombinant human baff, by Thermo Fisher Scientific (1 mentions)
Elisa plate reader, by Agilent Technologies (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Low melting point agarose, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Puromycin, by Merck Group (1 mentions)
Ez ecl, by Sartorius (1 mentions)
Superfect, by Qiagen (1 mentions)
Histostain sp broad spectrum kit, by Thermo Fisher Scientific (1 mentions)
Anti caveolin 1, by BD (1 mentions)
Orthovanadate, by Merck Group (1 mentions)
Rnaase free dnaase, by Promega (1 mentions)
8 protocols using mts proliferation assay kit
1
Assessing Cell Viability via MTS Assay
2
BAFF-R Signaling Pathway Analysis
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Major reagents included recombinant human BAFF (310–13, Peprotech, Rocky Hill, NJ, USA);BAFF-R antibody for flow cytometry and western blot (14–9117, eBioscience, San Diego, CA, USA); BAFF-R Fc chimera (1162-BR-050, R&D Systems, Minneapolis, MN, USA); phospho-Akt (2965) and Akt antibodies (9272, Cell Signaling Technology, Danvers, MA, USA); phospho-Erk (Thr202/Tyr204) (9106) and Erk antibodies (9102, Cell Signaling Technology); phospho-MAPKp38 (Thr180/Tyr182) (9211) and MAPKp38 antibodies (9212, Cell Signaling Technology); phospho-NF-κB p65 (Ser536) (3033) and NF-κB p65 antibodies (4764, Cell Signaling Technology); NF-κB p100 antibody(BS1247, Bioworld, China); Na+/K+-ATPase antibody (BS4259, Bioworld); carboxyfluoresceinsuccinimidyl ester (CFSE) (C34554, Life Technologies, Carlsbad, CA, USA); reverse transcription (RR014A) and real-time PCR kits (DRR036A, TAKARA, Shiga, Japan); TRIzol (15596–18) and DNaseI (AM2235, Life Technologies); protein extraction buffer (P0013, Beyotime, China); MTS Proliferation Assay kit (G3582, Promega, Madison, WI, USA); and a membrane protein extraction kit (89842, Pierce, Thermo Scientific, Waltham, MA, USA).
3
Cellular Viability and Proliferation
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Cellular viability was examined by counting the viable cells using trypan blue dye exclusion, and cellular proliferation was measured using an MTS proliferation assay kit (Promega, Madison, WI, USA). For the MTS assay, cells were plated on 96-well tissue culture plates at 5 × 104/mL in a total volume of 100 μL with the indicated agents and assayed according to the manufacturer's instructions. The absorbance at 490 nm was expressed as a relative value of the control culture.
4
BAFF-Induced Cell Proliferation Assay
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After serum starvation, cells were cultured in medium with 2 % FBS containing vehicle (PBS + 0.02%BSA) or variable concentration of BAFF protein for 48 h. Cell proliferation was measured with a MTS Proliferation Assay kit (Promega) based on the reaction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) in metabolically active cells. Total cell numbers were also manually counted and dead cells were excluded with eosin red staining.
For the CFSE assay, the cells were suspended at a concentration of 2 × 106 cells/mL and incubated with 5 μM CFSE for 5 min at 37 °C and then subjected to extensive washing with PBS. Afterwards, the cells were cultured with BAFF as indicated for 48 h then analyzed by flow cytometry. For the time course analysis of cell proliferation, cell proliferation was measured with the MTS Proliferation Assay kit at time points of 0, 6, 12, 24, 36, 48 and 72 h after administration of 20 ng/mL BAFF.
For the CFSE assay, the cells were suspended at a concentration of 2 × 106 cells/mL and incubated with 5 μM CFSE for 5 min at 37 °C and then subjected to extensive washing with PBS. Afterwards, the cells were cultured with BAFF as indicated for 48 h then analyzed by flow cytometry. For the time course analysis of cell proliferation, cell proliferation was measured with the MTS Proliferation Assay kit at time points of 0, 6, 12, 24, 36, 48 and 72 h after administration of 20 ng/mL BAFF.
5
Analyzing YAP5SA Impact on Cell Viability
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To evaluate the impact of YAP5SA overexpression on cell viability, EPO-CHO cells were seeded and grown in 96-well plates until they reached approximately 50% confluency. After 24, 48, and 72 h of transfection, a colorimetric cell viability assay was performed using the MTS proliferation assay kit (Promega, USA). To perform the assay, combining the MTS and PMS components beforehand is necessary, followed by an incubation period of 1 to 4 h. In brief, 100 µl of supernatant was removed from each well, and then 100 µl of fresh medium and 20 µl of the MTS-PMS reagent were added to allow for colorimetric evaluation of viable cells. The plates were subsequently read using an ELISA plate reader (Biotek; Germany), and the obtained data were documented.
6
Evaluating C3H10T1/2 Cell Proliferation
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C3H10T1/2 cells were plated on 96-well cell culture plates (2 × 104 cell/well) in 100 μL of medium and grown for 24 h at 37 °C. Thereafter, RSG 1 µM was added with or without EPA 10 µM and/or DHA 10 µM and the cells were incubated for another 72 h. Cell proliferation was evaluated using an MTS proliferation assay kit (Promega Inc., USA)57 (link).
7
Caveolin Expression Analysis Protocol
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The following reagents and antibodies were purchased from the sources indicated: 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) (Enzo, NY, USA), BCA protein assay kit (Pierce, Rockford, USA), DMEM-F12 (Gibco-BRL, Paisley, UK), Fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), Histostain-SP Broad Spectrum kit (Invitrogen, Carlsbad, USA), Penicillin, Streptomycin (Gibco-BRL, Paisley, UK), Puromycin (Sigma, USA), Superfect (Qiagen, Valencia, USA), Orthovanadate (Sigma, USA), Antipain, Benzamidine, Leupeptin, Phenylmethyl-sulphonylfluoride (Calbiochem, Germany), EZ-ECL (Biological Industries, Kibbutz Beit Haemek, Israel), RNAase-free DNAase (Promega, Madison, USA), TriZOL (Invitrogen, Carlsbad, USA), M-MLV Reverse Transcriptase (Promega, Madison, USA), Brilliant II SYBR Green Master Mix qPCR kit (Stratagene, USA), MTS® Proliferation Assay Kit (Promega, Madison, USA), low melting point (l.m.p) agarose (Invitrogen, Carlsbad, USA), polyclonal antibodies anti-caveolin-1 (BD, NJ, USA), anti-β-actin and anti-cytokeratin (Sigma, USA), monoclonal antibody anti-caveolin-2 (Santa Cruz Biotechnology, CA, USA), secondary antibody Goat anti-rabbit IgG coupled to horseradish peroxidase (Chemicon-Millipore, Billerica, USA).
8
Evaluating C3H10T1/2 cell proliferation
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C3H10T1/2 cells were plated on 96-well cell culture plates (2x10 4 cell/well) in 100mL of medium and grown for 24 hours at 37°C. Thereafter, RSG 1µM was added with or without EPA 10µM and/or DHA 10µM and the cells were incubated for another 72 hours. Cell proliferation was evaluated using an MTS proliferation assay kit (Promega Inc., USA) 55 (link) .
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