The effect on cell viability of the liposomes used for encapsulation was evaluated on human fibroblast MRC-5 (ATCC® CCL-171™) by MTT assay (Roche), following product instructions. Cells were treated for 24 h with different concentrations of HC, L or LHC and the viability was assessed by MTT assay (Roche). The HC 2000 and 10,000 µg/mL was used as positive control, as previously reported [9 (link),10 (link)]. The regenerative properties of the LHC were evaluated by scratch test assay [14 (link)], in comparison with the HC snail mucus. Briefly, the human fibroblast cell line MRC-5 was cultured in 24 wells until the formation of a cellular monolayer and then an artificial scratch was performed with a pipette tip on the culture surface. The cells were then treated with 2 mg/mL of HC Snail Mucus, water for the untreated control or LHC and the percentage of cellular migration has been evaluated after 4 h and 24 h of incubation at 37 °C.
Mtt assay
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The MTT assay is a colorimetric method used to measure the activity of enzymes that reduce the tetrazolium dye MTT to its insoluble formazan, creating a purple color. This reaction can be used to assess cell viability and proliferation.
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Lab products found in correlation
96 well plate, by Corning (5 mentions)
Microplate reader, by Bio-Rad (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Facscalibur, by BD (4 mentions)
Cell death detection elisa kit, by Roche (3 mentions)
Enspire multimode plate reader, by PerkinElmer (3 mentions)
Dtx880, by Beckman Coulter (2 mentions)
Imark microplate reader, by Bio-Rad (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Imt 2, by Olympus (2 mentions)
Xf24 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Clickredu solution, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
179 protocols using mtt assay
1
Evaluating Liposome Encapsulation Effects on Fibroblast Viability
2
Cell Viability Assay for Compound Screening
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Cells were seeded in 96-well plates at 2000 cells per well (optimum density for growth) in a total volume of 100 μL of medium containing 2% fetal bovine serum. Serially diluted compounds in 100 μL of medium were added to the cells 12 h later. After 72 h of incubation, cell viability was assessed by an MTT assay (Roche) according to the manufacturer's instructions.
3
CBD Effects on Cell Viability
4
MTT and Annexin V Assay Protocol
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In all, 5×103 HMEC or HUVEC cells were plated on 96 multiwells, and every 24 h cell growth was evaluated by MTT assay (Roche) according to the manufacturer’s instructions. For Annexin V affinity assay, 5×105 cells were collected, washed in PBS and labeled with Annexin V (BD Biosciences) for 20 min. Then, 2 μl of propidium iodide (1 mg/ml) (Sigma-Aldrich) were added. Annexin V emission was detected in the green channel (525 nm) and propidium iodide in the red channel (575 nm) on a FACSCalibur (BD Biosciences) using Cell Quest Pro Software (BD Biosciences).
5
MTT Assay for Cell Viability
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Cell viability was determined by MTT assay (11465007001; Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Briefly, the cells were cultured with 100 μl of medium per well in 96-well microplates. Following the addition of 10 μl of the MTT labeling reagent (final concentration, 0.5 mg/ml) to each well, the plates were incubated for 4 h in a humidified atmosphere at 37°C and 5% CO2. The plates were incubated overnight with 100 μl of the solubilization solution in each well. For the formation of purple formazan crystals, proportional to the number of metabolically active viable cells, the absorbance was measured using a microplate reader (DTX880; Beckman Coulter, Inc., Brea, CA, USA) at a wavelength of 570 nm.
6
Assessing CAG Cytotoxicity in hAMSCs
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CAG cytotoxicity for hAMSCs was assessed by the MTT assay (Roche Life Science), which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide to formazan crystals by living cells, which process determines the mitochondrial activity that can be photometrically detected (492 nm). The hAMSCs were seeded at 1 × 104 cells/cm2 in flat-bottomed 96-well plates, in 100 μL of basal culture medium (DMEM, low-glucose, Carlo Erba, Milan, Italy) with 2 mM L-glutamine, 1% pen/strep and 10% Fetal Bovine Serum. After 24 h, cells were cultured in different conditions: (1) basal medium and (2) basal medium supplemented with different concentrations of CAG (0.1/0.1/1/10/25 μM). To obtain CAG solutions at micromolar concentrations the CAG–DMSO stock solution has been highly diluted in basal medium (1:800–1:2000). The metabolic activity was then assessed by MTT conversion for 24 and 48 h. A blank control comprising the only medium was also included. For each measurement, three replicates per condition were included.
7
Cell Viability Evaluation via MTT Assay
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Cell viability was determined by the MTT assay (Roche Diagnosis, Indianapolis, IN, USA). Briefly, cells plated in 96-well plates (3000 cells/well) were treated with a siCDC5L or the NSP siRNA. Cell viability was determined 48 h after treatment.
8
Antiproliferative Effects of Dyes
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The antiproliferative effects of Methyl Orange, Alizarin Yellow, Lithol Rubine, Butter Yellow, Methyl Red and Sudan I were examined by using the MTT assay (Roche Diagnostics). The assay is based on the activity of metabolically active cells to cleave the MTT yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) to purple formazan crystals. Tests were conducted with 4,000 cells/well, plated in 200 µl media in 96-well plates, with four replicates. Cells proliferation was quantified 3 days and 7 days after treatment. To each well 10 µl MTT reagent was added, incubated for 4 h at 37 °C. Cells were then lysed by adding 100 µl solubilization buffer. The experiments were repeated 3 times in the same conditions. Each experiment had its own set of control. Optical density (OD) was measured using a spectrophotometer at 595 nm and relative cell viability was expressed as percentage of untreated control cultures.
9
Antiviral Potency of Compound 2 Against Influenza
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A549 cells were infected with A/WSN/33 and A/Vietnam/1203/04 at MOI 0.01, or with A/Panama/99 at MOI 0.1 in the absence or presence of compound 2 at concentrations depicted in the figures. Supernatants were collected from each condition 24 h post-infection and viral particles were tittered by plaque assay as following: MDCK cells were seeded in 6-well plates to reach confluency the next day. At confluency, ten-fold serial dilutions of each sample’s supernatant were diluted in PBS containing 100 units/mL Pen/Strep antibiotics, 0.2% BSA, 0.9 mM CaCl2, and 1.05 mM MgCl2. After infection with each dilution, cells were overlaid with a 1:1 mixture of 2X L-15 media and 2% Oxiod Agar (Final concentration of 1X L-15 media and 1% Agar). Plaques formed at 24 h for A/WSN/33 and A/Vietnam/1203/04, or 48 h for A/Panama/99 were counted and titers determined. Primary human bronchial epithelial cells were infected with A/WSN/33 at MOI 0.1 for 24 h in the absence or presence of compound 2 at depicted concentrations. Supernatants were subjected to plaque assays as described above.
Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions, concurrent with viral replication assay.
Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions, concurrent with viral replication assay.
10
EC Proliferation and Apoptosis Assay
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EC proliferation was induced by platelet‐derived growth factor (20 ng/mL) and was determined by MTT assay (Roche) and 5‐ethynyl‐2′‐deoxyuridine assay (EdU kit; RiboBio).16 EC apoptosis in cultured cells was induced by H2O2 (100 μmol/L) for 24 hours and measured by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (Roche).16
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