Cell proliferation was determined using the MMT assay (Sigma-Aldrich, St. Louis, MO, USA). Glioblastoma U87 cells were plated in 96-well microplate formats according to the manufacturer’s instructions. Cell lines were seeded in five replication wells at 5,000 cells/well and cultured for 0, 12, 24 or 48 h. Following MTT uptake for a duration of 4 h, cells were lysed in 150 μl dimethyl sulfoxide and absorbance was measured at 475 nm using a fluorescence microplate reader (Sunrise Remote, Tecan Austria GmbH, Grödig, Austria).
Mmt assay
Manufactured by Merck Group
Sourced in United States
The MMT assay is a laboratory test used to measure the metabolic activity of cells. It is a colorimetric assay that uses the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to quantify the number of viable cells in a sample. The assay measures the reduction of MTT to formazan, which is a purple-colored product, as an indicator of cellular metabolic activity.
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Lab products found in correlation
7 protocols using mmt assay
1
Proliferation Assay for Glioblastoma Cells
2
3D-Recovered Cell Cytotoxicity Assay
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For drug assessment, 3D-recovered cells were seeded in 96-well plates at a density of 80,000 cells/cm2. Cells were allowed to recover for 3 days and then treated with the following schedules: epirubicin 2 µg/ml plus ifosfamide 100 µM or trabectedin 17 ng/ml, according to the plasma peak levels of each compound (Highley et al., 2015 (link)). Medium was changed after 48 h and replaced with fresh complete DMEM. Survival percentages were assessed after a 24 h washout by MMT assay (Sigma-Aldrich) in accordance with the manufacturer's instructions.
3
Cell Viability Assay Protocol
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The cells were cultured on the substrates for 48 h for acute toxicity, and for 2 h to 21 days for chronic toxicity. Cell viability was measured by MMT assay (Sigma, St. Louis, MO, USA). The tetrazolium ring is cleaved by mitochondrial dehydrogenase enzymes to form a purple precipitate. MTT (0.5 mg/mL) was added to wells in high-glucose DMEM without phenol red. After 1 h incubation, the purple precipitate was dissolved in a 1:1 solution of isopropanol and dimethyl sulfoxide (DMSO). The absorbance of the solution was recorded at 570 nm (SpectraMax spectrophotometer, Molecular Devices, Sunnyvale, CA, USA).
4
Drug Combination Cytotoxicity Assay
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10,000 cells/well were seeded in 96-well plates. Cells were allowed to recover for 3 days before treatment. The regimens were selected on the basis of the peak plasma concentration of each drug obtained from pharmacokinetic clinical data; ifosfamide (IFO)100 µm,24 ,25 (link) epirubicin (EPI) 3.4 µm,26 (link),27 (link) EPI 3.4 µm plus IFO 100 µm, and trabectidin 2.2 × 10-5 µm.28 (link) Survival percentages were assessed by the MMT assay (Sigma Aldrich) after 72 h of drug exposure according to the manufacturer’s instructions. The experiments were performed twice.
5
Cytotoxicity Evaluation of Chemotherapies
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For drug assessment 10,000 cells were seeded per well in 96-well plates. Cells were allowed to recover for three days and then treated. Doses of ifosfamide, epirubicin and eribulin were selected on the basis of plasma levels from pharmacokinetic clinical data. As ifosfamide is used at high doses for the treatment of STS patients, it was administered to the primary culture at the concentration of 100 µM [25 (link),26 (link)]. Epirubicin 2 µg/mL, clinical studies have indicated that the Cmax of epirubicin is between 2 and 3.7 µg/mL [27 (link),28 (link),29 (link)]; epirubicin 2 µg/mL plus ifosfamide 100 µM; eribulin 371 ng/mL which corresponds to the plasma peak concentration in patients with solid tumors [30 (link)]. Percentages of survival were assessed by MMT assay (Sigma-Aldrich) after 72 h of drugs exposure, following the manufacturer’s instructions as previously described [31 (link)]. Two independent experiments were performed.
6
Evaluating Osteoclast-Mediated Drug Efficacy
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MCF7 and SCP2 cells were seeded at a concentration of 4000 cells per 96-well, and treated with drugs as reported for osteoclastogenesis assays. To evaluate the osteoclast effect on drug efficacy, cells were indirectly conditioned with/without osteoclast CM and GFs. Percentages of survival were then assessed by MMT assay (Sigma Aldrich) according to the manufacturer’s instructions. MTT was performed also on SCP2 directly co-cultured with osteoclasts.
7
Cytotoxicity Evaluation of Liposomal Drugs
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Cells were cultured in monolayer cultures or in 3D scaffolds for 24 h before exposure to drugs. Drug regimens were selected according to the plasma peak concentration of epirubicin from pharmacokinetic clinical data, 3.4 µg/ml46 (link) (which correspond for lipo-EPI and lipo-EPI-LOX to 110 µg of lipid component per ml). For lipo-LOX and lipo-EPI-LOX treatment groups, the same concentration of theoretical LOX was used (0,11 µg of anti-LOX per ml). For lipo and lipo-LOX treatment groups, the same concentration of lipid vesicles was used (110 µg/ml). Cell viability percentage was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MMT) assay (Sigma Aldrich, St. Louis, MO, USA) after 72-h of drug exposure as previously reported47 (link),48 (link). The IC50 values were calculated from the non-linear regression of the dose-log response curves. Experiments were performed in triplicate.
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