Fibroblasts of the 3T3 type were seeded into 96-well cell culture plates at a concentration of 12,500 cells/mL and cultured for 48 h. Conditioned media from fresh A-PRF, lyophilized A-PRF, as well as the control, at either 5% or 20% of the total volume, were added to each well. Cell proliferation was quantified using an MTT colorimetric assay (Invitrogen, Thermo Fisher Scientific, Life Technologies Corporation, Eugene, Oregon, USA) at 1, 3, 5, and 7 days according to the manufacturer’s instructions. Briefly, at desired time points, 10 µL of an MTT reagent at a concentration of 5 mg/mL was added and incubated at 37 °C with 95% humidity for another 2 h. Subsequently, the solution part was discarded and 100 µL of dimethyl sulfoxide (DMSO) (Sigma® Life Science, St. Louis, MO, USA) was added to dissolve the dark blue crystals of MTT formazan. The spectrophotometric absorbance (optical density) of the obtained solution was read using a microplate reader at 570 nm. The experiment was performed in triplicate, with two independent experiments performed. Cell proliferation numbers were presented as a percentage, which was calculated using the following formula (Equation (1)):
Mtt colorimetric assay
Manufactured by Thermo Fisher Scientific
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The MTT colorimetric assay is a laboratory technique used to measure cell viability and proliferation. It involves the conversion of the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into purple formazan crystals by metabolically active cells. The absorbance of the formazan solution is then measured, which is directly proportional to the number of viable cells present in the sample.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Infinite 200 microplate reader, by Tecan (2 mentions)
Spectramax microplate reader, by Molecular Devices (1 mentions)
Mtt reagent, by Thermo Fisher Scientific (1 mentions)
C4859, by Merck Group (1 mentions)
Z vad ome fmk, by MedChemExpress (1 mentions)
96 well plate, by Avantor (1 mentions)
Biomek plate reader, by Beckman Coulter (1 mentions)
Matrigel, by Corning (1 mentions)
Transwell chamber, by Corning (1 mentions)
96 well flat bottomed plate, by Corning (1 mentions)
Infinite 200, by Tecan (1 mentions)
Brdu labeling solution, by Thermo Fisher Scientific (1 mentions)
14 protocols using mtt colorimetric assay
1
Fibroblast Proliferation Assay with A-PRF
2
Cytotoxicity Evaluation of Nanoparticles
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Cytotoxicity of the nanoparticles of the
amino lipids was investigated using MTT colorimetric assay (Invitrogen,
Carlsbad, CA). NIH3T3-GFP cells were seeded onto 12-well plates at
a density of 5 × 104 cells per well. For each carrier,
the lipid/psgRNA and lipid/pCas9 nanoparticle ratio was 1:1 and nanoparticles
had concentrations of 1 μg/well for each plasmid, which were
incubated with NIH3T3 cells for 8 h at 37 °C. Then, the transfection
media was replaced with fresh serum-containing media (10% serum) and
cells were allowed to grow until 72 h. NIH3T3-GFP cells were incubated
with 100 μL MTT reagent for 4 h, followed by an additional 4
h incubation with 1 mL SDS-HCl solution to dissolve formazan crystals
formed by the reduction of MTT by NAD(P)H-dependent enzymes in the
cells. The absorbance of each sample was measured at 570 nm using
a SpectraMax microplate reader (Molecular Devices, San Jose, CA).
Cellular viability was calculated by averaging the signal intensities
over three replicates and then normalizing the results relative to
the negative control data.
amino lipids was investigated using MTT colorimetric assay (Invitrogen,
Carlsbad, CA). NIH3T3-GFP cells were seeded onto 12-well plates at
a density of 5 × 104 cells per well. For each carrier,
the lipid/psgRNA and lipid/pCas9 nanoparticle ratio was 1:1 and nanoparticles
had concentrations of 1 μg/well for each plasmid, which were
incubated with NIH3T3 cells for 8 h at 37 °C. Then, the transfection
media was replaced with fresh serum-containing media (10% serum) and
cells were allowed to grow until 72 h. NIH3T3-GFP cells were incubated
with 100 μL MTT reagent for 4 h, followed by an additional 4
h incubation with 1 mL SDS-HCl solution to dissolve formazan crystals
formed by the reduction of MTT by NAD(P)H-dependent enzymes in the
cells. The absorbance of each sample was measured at 570 nm using
a SpectraMax microplate reader (Molecular Devices, San Jose, CA).
Cellular viability was calculated by averaging the signal intensities
over three replicates and then normalizing the results relative to
the negative control data.
3
MTT Assay for Cell Viability
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Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Invitrogen, Eugene, OR, USA). Cell viability was quantified by the amount of MTT reduction [56 (link)]. HepG2 and EA.hy926 cells were exposed to different concentrations of the cluster (0.1, 1, 10, 50, 100 and 250 µM) for 48 h. After treatment, cells were co-incubated with MTT (0.5 mg mL) for 4 h, and then solubilized with an acidified (HCl 0.04 N) isopropanol/dimethyl sulfoxide (DMSO) solution. Optical density was measured at 540 nm. All experiments were performed as triplicate. Data were expressed as percentage of survival cell, compared to the control.
4
Assessing Neuroblastoma Cell Viability
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Neuron viability was assessed using the MTT colorimetric assay (Invitrogen) as previously described
[31 (link)]. Briefly, SH-SY5Y human neuroblastoma cells were treated with 2 μM tau oligomers, monomers, or APFs. After incubation for 6 h at 37°C, the cells were assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay kit (Tox-1) (Sigma) according to the manufacturer’s directions. All measurements were made in triplicate. Statistical analyses were based on a two-way ANOVAs, which were performed using Origin-8 software.
[31 (link)]. Briefly, SH-SY5Y human neuroblastoma cells were treated with 2 μM tau oligomers, monomers, or APFs. After incubation for 6 h at 37°C, the cells were assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay kit (Tox-1) (Sigma) according to the manufacturer’s directions. All measurements were made in triplicate. Statistical analyses were based on a two-way ANOVAs, which were performed using Origin-8 software.
5
MTT Assay and Immunofluorescence Staining for Cell Proliferation
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A3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Invitrogen, USA) was used to assess cell growth. Briefly, cells were cultured in a 96-well plate. A 20 μL volume of MTT medium was added to the cells for 4 h at room temperature, and then 200 μL DMSO (Sigma, USA) was added. The optical density was set at 545 nm (Bio-Rad, USA). Cells grown with no PECAM EMPs served as a control. The percentage growth rate was calculated.
Cell proliferation was evaluated using immunofluorescence staining as described previously23 (link). Cells were cultured in 24-well plates and treated with 2.8×105/mL PECAM EMPs at 0, 3, 6, 12, and 24 h. After treatment, the cell culture slides were incubated with the Ki67 monoclonal antibody (TA500265, OriGene, USA) at 4°C overnight. After washing, cells were incubated with cyanine 5-conjugated goat anti-mouse IgG (Thermo, USA) for 2 h at room temperature and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for 5 min from light and detected by a fluorescence microscope (Axioshop40, Carl Zeiss, Germany). All experiments were repeated three times.
Cell proliferation was evaluated using immunofluorescence staining as described previously23 (link). Cells were cultured in 24-well plates and treated with 2.8×105/mL PECAM EMPs at 0, 3, 6, 12, and 24 h. After treatment, the cell culture slides were incubated with the Ki67 monoclonal antibody (TA500265, OriGene, USA) at 4°C overnight. After washing, cells were incubated with cyanine 5-conjugated goat anti-mouse IgG (Thermo, USA) for 2 h at room temperature and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for 5 min from light and detected by a fluorescence microscope (Axioshop40, Carl Zeiss, Germany). All experiments were repeated three times.
6
MTT Colorimetric Assay for Cell Viability
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Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Invitrogen Life Technologies, Carslbad, CA, USA), in which cell viability was quantified by the amount of MTT reduction. After GW-788388 treatment was performed, endothelial cells were co-incubated with anhydrous MTT 4 h and then solubilized with an isopropanol/ DMSO solution. The optical density value was measured at 540 nm.
7
Inhibition of Cell Death Signaling
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Cells were incubated with different combination of a pan-caspase inhibitor, Z-VAD(OMe)-FMK (20 μmol/L, #HY-16658, MedChemExpress, NJ, USA), a cIAP1/2 inhibitor, BV-6 (10 μmol/L, #HY-16701, MedChemExpress) and an inhibitor of protein synthesis, cycloheximide (CHX) (0.5 μg/mL, #C4859, Sigma-Aldrich) 1 h before TNF-α stimulation (20 ng/mL, #315-01A, PeproTech, Neuilly-sur-Seine, Paris). Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTT) colorimetric assay (Thermo Fisher Scientific), which measures cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH) to reduce the MTT to its insoluble formazan end product, which has a purple color. Cells were incubated with 0.5 mg/mL of MTT reagent (Thermo Fisher Scientific) for 2 h. Once MTT crystals were developed and controlled under light microscopy, they were dissolved in DMSO and quantified by measuring absorbance at 540 nm.
8
MTT Assay for Cell Viability
9
Limbal-Epithelial Cell Viability Assay
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Human corneal limbal-epithelial cells were cultured in the presence of different SDP concentrations (0.2%, 0.4%, and 0.5% wt/vol) or PBS vehicle control, in a 96-well plate (VWR), at a cell seeding density of 3 × 103 cells/cm2. The cultures were then subjected to the MTT colorimetric assay (ThermoScientific) per manufacturer instructions at 12 hours post treatment, which was the duration when the greatest impact of wound healing was observed in vitro. Briefly, 50 μL MTT stock solution (5 mg/mL) was added to the cultures containing 500 μL fresh medium and incubated at 37°C in the dark for 4 hours. After the medium was aspirated, 200 μL dimethyl sulfoxide (Sigma-Aldrich Corp.) was added and mixed thoroughly to release the formazan. The absorbance of the resultant solution was recorded at 540 nm by using a Biomek plate reader (Beckman Coulter, Jersey City, NJ, USA). Wells containing culture media, without cells, were set up as negative controls.
10
Assessing HAMLET's Cytotoxic Effects
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We used the MTT colorimetric assay (obtained from Thermo Fisher Scientific and Carl Roth in Steinheim, Germany and Karlsruhe, Germany) to assess cell viability and the cytotoxic effects of HAMLET over 48 h. We initially seeded cells into 96-well plates at densities appropriate for each cell line, ranging from 8000 to 20,000 cells per well. After 24 h, we treated the cultures with the HAMLET complex and incubated them for another 6 h. We then replaced the growth medium and allowed the cells to incubate for another 18 h before adding the MTT reagent. After incubation for 3–4 h at 37 °C, we dissolved the formazan crystals in dimethyl sulfoxide, measured the absorbance at 570/620 nm, and compared it with a control group.
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