The cell viability of transfected MKN-45 cells was assessed using the MTT Cell Proliferation Assay Kit (Beyotime, Shanghai, China) in line with the manufacturer’s guidance. Transfected MKN-45 cells (5 × 104 per well) were incubated with 10 μL of MTT solution (5 mg/mL prepared with PBS [pH 7.4]) at 37°C for 4 h. Then, stop solution was added to the cells and incubated at 37°C overnight. The optical density value was determined at 570 nm absorbance with the Thermo Scientific Microplate Reader Multiskan MK3 (Thermo Fisher Scientific).
Mtt cell proliferation assay kit
Manufactured by Beyotime
Sourced in China
The MTT Cell Proliferation Assay Kit is a colorimetric assay used to measure the metabolic activity of cells. It is used to quantify cell proliferation, viability, and cytotoxicity. The assay involves the conversion of a tetrazolium dye, MTT, into a colored formazan product by the mitochondrial enzymes of viable cells.
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Lab products found in correlation
Spectrometer reader, by Olympus (2 mentions)
Thermo scientific microplate reader multiskan mk3, by Thermo Fisher Scientific (1 mentions)
Caspase 3 activity kit, by Beyotime (1 mentions)
Spectramax absorbance reader, by Molecular Devices (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cck 8 assay, by Beyotime (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Cellrox oxidative stress reagent, by Thermo Fisher Scientific (1 mentions)
Cell culture flask, by Corning (1 mentions)
Hepg2 cell line, by Shanghai Cell Bank (1 mentions)
Puromycin, by Corning (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Ag1478, by Cayman Chemical (1 mentions)
Cay10657, by Cayman Chemical (1 mentions)
U0126, by Cayman Chemical (1 mentions)
Anti phosphotyrosine antibody, by Abcam (1 mentions)
Anti erbb2, by Cell Signaling Technology (1 mentions)
Anti erbb3, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
22 protocols using mtt cell proliferation assay kit
1
Cell Viability Assessment in MKN-45 Cells
2
Cell Proliferation and Caspase 3 Activity Assays
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Cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) assay according to the instructions of the MTT cell proliferation assay kit (Beyotime Biotechnology). The optical density was measured at 570 nm on a microplate reader (Bio-Rad 550; Bio-Rad, CA, USA). The activity of Caspase 3 was determined using the Caspase 3 activity kit (Beyotime Biotechnology). Cell lysates were prepared after their respective treatment. The cells were then scraped, centrifuged, resuspended and lysed in lysis buffer according to the manufacturer's protocol. Assays were performed in 96-well microtiter plates by incubating 10 μl protein of cell lysate per sample in 80 μl reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM Nicotinamide adenine dinucleotide (NAD) and 10% glycerol) containing 10 μl 2 mM Caspase3 substrate (Ac-DEVD-pNA). Lysates were incubated at 37°C for 4 hrs. Absorbance of the samples was then measured using by a microplate reader (Bio-Rad 550; Bio-Rad) at 405 nm, according to the manufacturer's protocol.
3
Glioma Tissue Sampling and Cell Culture
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All specimens were from 43 patients provided by the Department of Neurosurgery, Shengjing Hospital of China Medical University from March 2010 to May 2012. All activities were approved by the review boards of our hospital, and informed consent was obtained from all participants. The tumors with at least 1 cm margin from the corresponding peri-cancerous tissues were obtained from all patients through surgical resection and further histologically proven to be gliomas. The patients include 29 men and 14 women (mean age: 51.3 ±4.6 years, age range: 47–66 years). All patients were naive to chemotherapy and radiotherapy before surgery, including 23 cases of astrogliomas (grade I–II), 11 of anaplastic gliomas (grade III), and 9 of glioblastomas (GBM, grade IV). Human glioblastoma cell line U251 was obtained from Biological Sciences Cell Resource Center (China). Real-time polymerase chain reaction (PCR) reagents were from Takara Bio (China), siRNA-FUBP1, siRNA-control and TransMessenger from Invitrogen (Carlsbad, CA, USA), and DDP from Sigma (USA). The MTT Cell Proliferation Assay Kit was from Beyotime Company (Shanghai, China), and Annexin V Cell Apoptosis Assay Kit was from Biosea Company (Beijing, China). The PCR primers of the FUBP1 gene were synthesized by Sangon Biotech (Shanghai, China).
4
miR-17-5p Regulation of Cell Proliferation
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Cells were seeded in a 96-well plate at a density of 1 × 10 3 cells per well, and transfected with miR-17-5p mimics, miR-17-5p inhibitor and miR-NC as described above. Cell proliferation was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium Bromide (MTT) assay in keeping with instructions of the MTT cell proliferation assay kit (Beyotime Institute of Biotechnology). Optical density (OD) was measured at 490 nm on a microplate reader (Bio-Rad 550, USA). All experiments were performed in triplicate and repeated at least three times.
5
MTT Assay for Cell Proliferation
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A total of 3 × 104 cells/well were seeded in 96-well plates. After treatment with various concentrations of BTZ (S1013, Selleck) at 37°C for 24 h, cell proliferation was measured using an MTT cell proliferation assay kit (Beyotime, Shanghai). Absorbance was measured at 490 nm using a SpectraMax Absorbance Reader (Molecular Devices, San Francisco, CA, USA). The experiment was repeated three times.
6
MTT Cell Proliferation Assay Protocol
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The MTT Cell proliferation Assay Kit (Beyotime, Shanghai, China) was used to detect cell proliferation capacity. Generally, cells with different transfections were grown in 96-well plates (2000 cells/well) for the desired time (24, 48 and 72 h). Four hours before the end time point of culture, 10 μL MTT solution was added into each well, incubating for another 4 h at 37°C. Afterwards, 100 μL formazan solving liquid was added into each well until the formazan was fully dissolved. The absorbance at 490 nm was recorded using the iMark microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were repeated in triplicate.
7
Evaluating siRNA Efficacy against EV71 Infection
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RD cells were seeded in a 24-well plate and then transfected with 60 nM siRNA. After 24 h, the cells were infected with 0.01 MOI of strain EV71-2006-52-9. At one day postinfection, the cells were trypsinized and resuspended in DMEM. The resuspended cells (100 μl) were transferred to a 96-well plate and grown for another 24 h. To assess the viability of the RD cells transfected with siRNA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed strictly according to the manufacturer’s instructions of the MTT Cell Proliferation Assay Kit (Beyotime, China). Morphological changes in the RD cells transfected with siRNA were evaluated with inverted microscopy at 48 h postinfection. All assays were performed in triplicate in three independent experiments.
8
Cell Proliferation Evaluation by MTT Assay
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CRC cell proliferation was evaluated by MTT Cell Proliferation Assay Kit (Beyotime, Shanghai, China). In brief, 2×103 cells/100μl/well were added to the wells of 96-well plates in triplicate. After transfection, 10 μl MTT (5 mg/mL) was added into the wells at each point in time. After the cells were cultured for 4 h at 37°C, 100 μl of Formazan dissolved solution was added into the wells. A microplate reader (Bio-Rad, Hercules, CA, USA) was used to detect the optical density at 570 nm (OD570 nm). MTT assay was repeated three times.
9
Cell Proliferation Assays for Cancer Cell Lines
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Twenty-four hours after transfecting METTL3 expression plasmids, HCCLM3 or HepG2 cells were seeded at 5 × 103 cells per well in 24-well plates and cultured for 5 days. Cell numbers were counted every day using a hemocytometer.
CCK-8 assays (Beyotime, Shanghai, China) were performed to determine cell proliferation. Briefly, 100 μL of 2 × 103 cells was seeded into each well of the 96-well plate and incubated in a 5% CO2 incubator at 37 °C for 0, 24, 48, and 72 h. Next, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance was measured at 450 nm using a spectrophotometer.
MTT assays were performed using an MTT Cell Proliferation Assay Kit (Beyotime, Shanghai, China). Briefly, 100 μL of 5 × 103 cells was seeded into each well of the 96-well plate and incubated for 0, 24, 48, and 72 h at 37 °C, and then 10 μL of MTT stock solution was added for another 4 h of incubation. The 96-well plate was then foil wrapped after adding 100 μL of DMSO solution, followed by shaking on an orbital shaker for 15 min to fully dissolve the MTT formazan. Absorbance was measured at 490 nm using a spectrophotometer.
CCK-8 assays (Beyotime, Shanghai, China) were performed to determine cell proliferation. Briefly, 100 μL of 2 × 103 cells was seeded into each well of the 96-well plate and incubated in a 5% CO2 incubator at 37 °C for 0, 24, 48, and 72 h. Next, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance was measured at 450 nm using a spectrophotometer.
MTT assays were performed using an MTT Cell Proliferation Assay Kit (Beyotime, Shanghai, China). Briefly, 100 μL of 5 × 103 cells was seeded into each well of the 96-well plate and incubated for 0, 24, 48, and 72 h at 37 °C, and then 10 μL of MTT stock solution was added for another 4 h of incubation. The 96-well plate was then foil wrapped after adding 100 μL of DMSO solution, followed by shaking on an orbital shaker for 15 min to fully dissolve the MTT formazan. Absorbance was measured at 490 nm using a spectrophotometer.
10
Cell Proliferation Assay with miR-17-5p
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Cells were seeded in a 96-well plate at a density of 1×103 cells per well, then cells were transfected with miR-17-5p mimics and miR-NC as described above. Cell proliferation was determined by MTT assay according to the instructions of a MTT cell proliferation assay kit (Beyotime Biotechnology, Jiangsu, China). The optical density was measured at 570 nm on a microplate reader (Bio-Rad 550, USA).
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