Specimens with the dimensions of 1×1 cm were sterilized by submerging in ethanol 100%, ethanol 70%, and ethanol 50% for 1 hour, 30 min, and 30 min, respectively, followed by washing with sterilized PBS[28 ]. The sterile scaffolds (3±0.5 cm2/mL of culture medium) were placed in a 12-well tissue culture plate, washed three times with fresh culture medium and incubated in fresh culture medium at 37°C for 1, 3, and 14 days to get scaffold extract. As the control group, serum-supplemented culture medium (DMED) at the same condition was used. Then the cells were seeded into 96-well plates at a density of 1×104 cells/mL. The culture medium (100 μL) of cells in 96-plates was replaced with obtained extracts as so the control group medium. After 24 h of incubation, the viability of each group was assessed using MTT (Sigma-Aldrich, USA). The medium was removed, and 100 μL of a 0.5 mg/mL MTT solution was added to each well and incubated at 37°C for 4 h. Finally, MTT solution was removed gently and 150 μL isopropanol (Sigma-Aldrich, USA) was added to each well to dissolve the MTT formazan purple crystals. The absorbance of the solutions was measured at 545 nm using an ELISA Reader (Stat Fax-2100, USA). The normalized relative viability or cell growth (%) was calculated from the following equation:
Methyl thiazolyl tetrazolium (mtt)
Manufactured by Merck Group
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MTT is a colorimetric assay used to measure cell metabolic activity. It is a lab equipment product developed by Merck Group. MTT is a tetrazolium dye that is reduced by metabolically active cells, producing a colored formazan product that can be quantified spectrophotometrically.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (1605 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (457 mentions)
Microplate reader, by Bio-Rad (286 mentions)
Microplate reader, by Agilent Technologies (204 mentions)
Microplate reader, by Thermo Fisher Scientific (203 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (137 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (117 mentions)
Microplate reader, by Molecular Devices (106 mentions)
Multiskan fc, by Thermo Fisher Scientific (101 mentions)
Streptomycin, by Thermo Fisher Scientific (93 mentions)
Penicillin, by Thermo Fisher Scientific (85 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (83 mentions)
96 well plate, by Corning (83 mentions)
Propidium iodide, by Merck Group (82 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (82 mentions)
Fetal bovine serum (fbs), by Merck Group (74 mentions)
Elx800, by Agilent Technologies (73 mentions)
Microplate reader, by Tecan (68 mentions)
Lipopolysaccharides (lps), by Merck Group (66 mentions)
Trypsin, by Thermo Fisher Scientific (65 mentions)
9 566 protocols using methyl thiazolyl tetrazolium (mtt)
1
Evaluating Cell Viability of Scaffold Extracts
2
Assessing Mitochondrial Activity of BHK-21 Cells
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Mitochondrial activity of BHK-21 cells seeded
onto the developed biocomposite films was assessed by enzymatic conversion
of tetrazolium dye MTT (Sigma-Aldrich) after the time intervals of
8, 16, 24, 48, and 72 h. MTT reagent was prepared by dissolving 5
mg of MTT in 1 mL of phosphate-buffered saline (PBS). After each interval,
10 μL of MTT reagent was added to every well and incubated for
3 h at 37 °C in a humidified atmosphere. Later, the MTT reagent
was removed from the wells and 100 μL of dimethyl sulfoxide
(Merck) was added to dissolve the formosan crystals. Absorbance were
measured using plate reader (Thermo Fischer Scientific) at 570 nm
with reference wavelength of 650 nm.
onto the developed biocomposite films was assessed by enzymatic conversion
of tetrazolium dye MTT (Sigma-Aldrich) after the time intervals of
8, 16, 24, 48, and 72 h. MTT reagent was prepared by dissolving 5
mg of MTT in 1 mL of phosphate-buffered saline (PBS). After each interval,
10 μL of MTT reagent was added to every well and incubated for
3 h at 37 °C in a humidified atmosphere. Later, the MTT reagent
was removed from the wells and 100 μL of dimethyl sulfoxide
(Merck) was added to dissolve the formosan crystals. Absorbance were
measured using plate reader (Thermo Fischer Scientific) at 570 nm
with reference wavelength of 650 nm.
3
MTT Assay for Cell Viability
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Cells were grown and infected with HIF-2α shRNA or negative control lentivirus for 72 h and then seeded for cell viability assay. For the methyl thiazolyl tetrazolium (MTT) assay, 20 mL MTT (5 mg/mL, Sigma) was added to each well of the 96-well plates, incubated for 4 h, and then the growth medium was removed and 100 mL dimethyl sulfoxide was added to the wells to dissolve the MTT crystals. Next, the optical densities were measured using an automated spectrophotometric plate reader at a wavelength of 570 nm. The percentage of cell growth was calculated using the formula: % control = ODt / ODc x 100, where ODt and ODc are the optical densities for transfected and vector control cells, respectively.
4
Cytotoxic Effects of Test Agents on K562 Cells
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The cytotoxic effects of the test agents on the viability of the K562 cells was evaluated by the thiazolyl blue tetrazolium bromide (MTT) colorimetric assay, as well as Trypan blue dye exclusion test. For both experiments, the cells were seeded into 12-well plates and treated with FIS at concentrations of 10, 20, 30, 40, 50 µM for 24 or 48 h. After the indicated treatment period, the cells were incubated in the MTT working solution, which was prepared by diluting the MTT stock solution [made by dissolving 5 mg of MTT (Sigma-Aldrich) in 1 ml of PBS] with the culture medium without phenol red (HyClone, Thermo Fisher Scientific) at a ratio of 1:9. Following 3 h of incubation in the CO2 incubator at a standard condition, the resulting formazan crystals were dissolved in isopropanol (POCH, Poland) and the absorbance was read at 570 nm using a spectrophotometer (Spectra Academy, K-MAC). Following Trypan blue staining (3 min at room temperature), at least 100 cells per data point were counted under an Eclipse E800 light microscope (Nikon Corp.) and blue stained cells were considered dead. The cell viability was calculated assuming the absorbance of control cells as 100%.
5
MTT Assay for Cell Viability
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Cell suspension was diluted and inoculated into a 96-well plate with a density of 3 × 10 3 per well. Six parallel wells were set for each group. After cell confluence reached 80%, cells were grouped based on the above arrangements. After re-oxygenation, cells were added with 20 µL of PBS-diluted 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; 5 mg/mL, 0.5% MTT; Sigma-Aldrich Chemical Company) in a 37°C incubator for 4 h. Then, the MTT was aspirated, and each well was added with 150 µL of dimethyl sulfoxide (Sigma-Aldrich Chemical Company) in a shaking bed for 10 min. A microplate reader was used to measure the optical density (OD) with the wavelength of 570 nm. Each experiment was conducted for three times to obtain the average OD value. Cell survival rate = (OD value in experimental group -OD value in control group)/OD value in control group. Each experiment was conducted for three times to obtain the average value.
6
Cytotoxicity Screening of Compounds in HepG2 Cells
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HepG2 cells were purchased from American Type Culture Collection. The cells were cultured in Eagle’s Minimum Essential Medium (Ozyme, France) supplemented with 10% fetal bovine serum, 1X non-essential amino acids, 100 units/mL penicillin and 10 mg/mL streptomycin (Dutscher, Brumath, France). Cultures were kept under a CO2/air (5%/95%) humidified atmosphere at 37 °C. Prior to the experiment, cells were seeded in 96-well culture plates at a density of 0.1 × 106 cells per well. After 24 h of incubation, the culture medium was refreshed and 100 µL of the test compounds or DMSO (0.1%) were added. Compounds were tested at 4 concentrations (1–30 µM) in triplicate. For the MTT assay [25 (link)], after 24 h of treatment, cells were incubated with 50 µL MTT (0.5 mg/mL, Sigma Aldrich, city, France) at 37 °C for 2 h. Plates were centrifuged, MTT was removed and 100 µL DMSO was distributed per well. The absorbance at 570 nm was measured using microplate reader (brand). Cell viability was expressed as percentage of cell viability compared to controls (DMSO, 0.1%).
7
Cell Viability Evaluation by MTT Assay
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The MTT assay was used for cell viability evaluation. Cells were seeded to 96-well plates with 2000 cells/well and incubated for 1, 2, 3, 4, and 5 d. In the MTT assay, in each group, 20 μl 5 mg/ml, MTT (Sigma–Aldrich, U.S.A.) solution was added to each well and then the cells were continue to incubate at 37°C for 4 h. After that, the MTT solution was removed, and 200 µl of DMSO was added for each well. After 15 min incubating in 37°C, the plate was measured by a microplate reader at 490 nm (BioTek Instruments, U.S.A.).
8
MTT Cell Proliferation Assay
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The cell proliferation and cytotoxicity assay was carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, USA) as follows: COS-7 cells were cultured in 96-well plates (8 × 103 cells per well) for 24 h, and then incubated with different concentrations (0, 2.5, 5, 10, and 20 µM) of DAMB-SA, DAMB-SAN for 24 h. The medium in each 96-well plate was removed completely and 100 µL of MTT mixed solution was added to each culture well, which consisted of 90 µL of culture medium and 10 µL of MTT solution (5 mg mL−1). After incubation at 37 °C and in 5% CO2 atmosphere for 4 h, 100 µL of DMSO was added to each well after the 100 µL MTT mixed solution removed. Finally, OD was measured using an enzyme mark instrument (Tecan, infinite F50, Switzerland), at an optical absorbance of 570 nm. Five replicate measurements were obtained for each sample (n = 5). The survival rate of cells was determined by dividing the cell viability of the cells incubated with DAMB-SA, DAMB-SAN by the cell viability of the control group performed in the absence of DAMB-SA, DAMB-SAN.
9
Cytotoxicity Evaluation of PEI-alginate/siRNA Nanocomplexes
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Cytotoxicity of PEI-alginate/siRNA nanocomplexes was examined by MTT (methylthiazolyldiphenyl-tetrazolium bromide; Sigma, CA, USA) assay. The cells were seeded in 96-well plate at density of 5 × 103 cells (150 μl medium). After incubation for 24 h, the cells were treated with the nanocomplexes containing negative control siRNA with different N/P ratios for 4 h. Then, the medium was replaced, the cells continued to be incubated for 20 h. The cells treated with PEI/siRNA complexes were taken as the control. After adding of MTT solution (5 mg/ml), the cells were incubated for 4 h. Following removal of the medium containing MTT, 150 μl dimethylsulfoxide (DMSO; Sigma, CA, USA) was added to dissolve formazan crystal formed by live cells. The absorbance was measured at 540 nm with an absorbance microplate reader (BIO-RAD, Hercules, CA, USA). The assay was repeated thrice. Cell viability was calculated by the equation: cell viability (%) = [OD540 (sample)/OD540 (control)] × 100%. Changes of cell viability in PEI-alginate/siRNA group were compared with PEI/siRNA group.
10
Evaluating Foxp3 Silencing Effects on B16F10 Cell Viability
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Briefly, 24 hours before transfection, 5×105 B16F10 wild-type (WT) cells were seeded in 96-well culture plates. Thereafter, B16F10 WT cells were transfected with small interference Foxp3 (siFoxp3) plasmids (siFoxp3-1, siFoxp3-2, and siFoxp3-3) and pEGFP-N2 (as a negative control). The complex PEI:DNA was performed as described previously. At 72 hours, 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed for cell viability analysis. The MTT solution was prepared by mixing 0.025 g of MTT (Sigma-Aldrich Co.) with 5 mL of phosphate-buffered saline (PBS) for a final MTT concentration of 5 mg/mL. Then, 20 µL of this solution was added to each well. The cells were incubated with the MTT solution at 37°C for 2–4 hours until a purple precipitate was visible, followed by removal of the medium and addition of 80 µL dimethylsulfoxide to each well. The samples were rocked for 10 minutes. The optical density was determined at 570 nm using a microplate autoreader (Microplate Autoreader EL311; BioTek Instruments Inc., Winooski, VA, USA). Data are presented as the percentage of relative cell viability ± standard error.
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