The MTT assay was applied to measure cell proliferation. The cell-laden hydrogels were washed in phosphate-buffered saline (PBS), followed by the addition of 200 μL of 0.1 mg/mL tetrazolium dye (Sigma-Aldrich, St. Louis, MI, USA) and incubation for 4 h at 37 °C. After incubation, the tetrazolium dye was removed. The reduced product of tetrazole, the purple formazan, was dissolved by DMSO and detected at 570 nm using a microplate reader (SpectraMax® M5, Molecular Devices LLC, Sunnyvale, CA, USA). The optical density was normalized to that measured for cells (cell density: 2 × 106 cells/mL) before mixing with hydrogels (100% viability). The viabilities were measured at the 1st, 2nd, 3rd and 7th day of culture. The medium without cells and cells in a blank well (tissue culture polystyrene (TCPS)) were used as control groups, where the MTT assay was performed for cells on 24-well plates (cell density: 3 × 104 cells/wells), and the viabilities were normalized to those measured for cells before the culture. The results were proven to be reproducible in at least three independent experiments. Through one-way analysis of variance, p-values < 0.05 indicated significant statistical differences.
Tetrazolium dye
Manufactured by Merck Group
Sourced in United States
Tetrazolium dye is a colorimetric reagent used in various laboratory applications. It serves as an indicator for cellular metabolic activity and viability. The dye undergoes reduction in the presence of metabolically active cells, leading to the formation of a colored formazan product that can be quantified spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Cisplatin, by Merck Group (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Versamax, by Molecular Devices (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
Gelatin, by Merck Group (1 mentions)
Lysozyme enzyme, by Merck Group (1 mentions)
Fluorescence plate reader, by Bio-Rad (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Elisa microplate reader, by Agilent Technologies (1 mentions)
Vacutainer cpt, by BD (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Ficoll, by Biowest (1 mentions)
13 protocols using tetrazolium dye
1
Measuring Cell Viability Using MTT Assay
2
Evaluating Cisplatin and 5-Aza-CdR Cytotoxicity
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Tetrazolium dye (MTT; Sigma-Aldrich, St. Louis, MO) assays were used to evaluate the inhibitory effect of cisplatin or 5-Aza-CdR on cell growth. Cells were seeded in 96-well plates, incubated for 24 hours, and treated with cisplatin alone or in combination with 500 nM 5-Aza-CdR for 72 hours at 37°C. After drug treatment, MTT solution was added to each well and cells were incubated for 4 hours at 37°C before removal of the media. Cells were then treated with dimethyl sulfoxide (Sigma) and the absorbance of the converted dye in living cells was measured using a microplate reader (Versa-Max, Molecular Devices, Sunnyvale, CA) at a wavelength of 540 nm. Six replicate wells were used for each analysis and three independent experiments were performed.
3
Analyzing Cell Proliferation with MTT Assay
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In a 96-well flat-bottomed plate 1000 cells/100 μL of DMEM were seeded. Following serum starvation cells were stimulated with TGFβ (2 ng/mL) and MTT, a tetrazolium dye (Sigma-Aldrich) was added to each well after 1, 3, and 5 days of TGFβ stimulation. After adding MTT, plates were incubated at 37 °C for 4 h, finally 100 µL of DMSO was added to each well to solubilize the purple colored formazan crystals and absorbance was recorded at 560 nm on a microplate reader.
4
Lysozyme-Loaded Chitosan-Gelatin Hydrogel
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Chitosan (medium molecular weight, 75–85% degree of deacetylation), gelatin (from pork skin), NHS, lysozyme enzyme, and Tetrazolium dye for MTT test were purchased from Sigma Aldrich Company. Regarding the product information of lysozyme from chicken egg white for Molecular Biology (Catalog Number L7651, Sigma-Aldrich, USA), lysozyme activity was ≤ 40,000 units/mg protein. EDC was purchased from BioBasic Inc. (Canada). Allantoin was dedicated by Pars Hayan Co.
5
HepG2 Cell Proliferation Assay
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HepG2 cells (1×104 cells in 100 μl medium) from the different groups were added to 96-well plates and incubated for 24 hours under serum-starved conditions. The MTT cell proliferation assay tetrazolium dye (20 μl, 5 mg/ml, Sigma) was placed into each well and cultured for 4 h at 37°C in an incubator with 5% CO2. Then, 150 μl dimethyl sulfoxide (DMSO) (Sigma, St Quentin Fallavier, France) was added to each well, and cells were gently shaken for 10 min to dissolve the crystals. Absorbance was detected at 490 nm by a fluorescence plate reader (Bio-Rad, USA) on day 1, 2, 3, 4, and 5, respectively. Wells with medium only were used as the blank control and cell viability was confirmed by measuring the optical density (OD) ratio between the treated culture group and the untreated control group. The assays were performed in triplicate.
6
Cytotoxicity Assay under Normoxia and Hypoxia
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The cells were plated into 96-well plates (104/well) and incubated with various concentrations of GCV and/or 5-FC for 48 h under normoxic or 0.5% O2 conditions. The medium containing the drug was then replaced with fresh medium, and cells were cultured for an additional 48 h under the normoxic conditions. Tetrazolium dye (Sigma) was added and allowed to react for 2 h at 37°C. After dissolving in DMSO, dye conversion was read using an ELISA plate reader at 490 nm against 610 nm.
7
Cytotoxicity Assay for Anticancer Compounds
8
Cytotoxic Effects of BSO on PBMCs
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For cytotoxicity analysis, human blood obtained from Sulaimani Blood Bank was used. Peripheral blood mononuclear cells (PBMCs) were separated using a Vacutainer® (CPT™; BD, Franklin Lakes, NJ, USA) containing the cell separation medium with sodium citrate, following the instructions of the manufacturer. The cytotoxic effect of BSO on PBMCs was determined at several concentrations (25, 50, and 100 μg/mL) after 24, 48, and 72 hours of incubation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously described [28 (link)]. In brief, 20 µL of tetrazolium dye (Sigma-Aldrich Co., USA) was added to each well at a concentration of 5 mg/mL diluted in phosphate buffer saline (PBS), and the plates were incubated for 4 hours at 37°C. Afterwards, about 180 µL of the solution was dropped from each well and replaced with 100 µL dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., USA). The systems were well shaken using an electronic shaker and read using the ELISA microplate reader (BioTek, Germany). Finally, the obtained data were analyzed and plotted. The analysis was performed in triplicate, and DMSO was used as the negative control.
9
Astrocyte Metabolic Activity Assay
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Following in vitro overpressure exposure, astrocytes were assessed for metabolic (NADPH-dependent) activity by MTT assay. At 24 and 48 h post-overpressure, cell media was changed and supplemented with tetrazolium dye (Sigma-Aldrich Cat# M2128) at a final concentration of 0.25 mg/mL. Cultures were incubated at 37°C for 3 h with the tetrazolium before being dissolved in dimethyl sulfoxide. Samples were then transferred in triplicate to a 96-well plate and absorbance was read at 570 and 650 nm (background). Percent activity was calculated based on average optical density measurements for the sham samples.
10
Quantification of Bacterial Populations and Phages
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After serial dilutions, bacterial populations were determined by plating on selective antibiotic-LB agar plates (1.5% agar). Antibiotics were used at the following concentrations: kanamycin (50 μg/mL), chloramphenicol (20 μg/mL), and phleomycin (5 μg/ml). PFUs were determined by spotting 10 μl of serial dilutions of diluted feces on a lawn of the indicator bacteria in top agar (0.4% agar, 10 mM MgSO4). The indicator bacterial culture was fresh MD5 culture grown in LB containing 0.2% maltose. Latent bacteria were counted similarly but after elimination of free phage by centrifugation. CFUs and PFUs were counted after 12–16 hours of incubation at 37°C. The ability to use maltose was monitored in tetrazolium maltose (TM) indicator plates. Mal+ and Mal- clones respectively form white and red colonies on these plates. The TM medium was composed of tryptone (10 g/L), yeast extract (1 g/L), NaCl (5g/L), agar (16g/L), maltose (5 g/L) and tetrazolium dye (50 mg/L, Sigma).
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