Mtt cell proliferation assay kit

Doubling time was measured by counting trypsinized cells using a hemocytometer. Cells were trypsinized after reaching 80% confluency, at passages 1–5. Proliferation was determined using an MTT Cell Proliferation Assay Kit (Invitrogen, Waltham, MA). Briefly, cells were seeded in a 96-well plate, cultured for 10 days, incubated with MTT for 4 hours, and measured spectrophotometrically.

The cytotoxicity of tested extracts was measured by performing MTT assay using the MTT Cell Proliferation Assay Kit (Invitrogen, Waltham, MA, USA). The test is based on the ability of living cells to reduce orange tetrazolium salt by cellular dehydrogenases. to water-insoluble purple formazan crystals. Cells were seeded into 96-well plates in the concentration range from 1 × 10⁵ cells/mL to 2.5 × 10⁵ cells/mL depending on the used cell line. Stock solutions for cytotoxicity analysis were prepared by dissolving 50 mg of dry plant extracts in 1 mL of DMSO. For biological activity studies, extracts not purified by SPE were used. Tested extracts were added to the cell cultures when 70–80% of confluence was achieved in a wide range of concentrations (maximum concentration 125 µg/mL). MTT solution (4 mg/mL) was added to the culture after 48 h of incubation with tested extracts. Following 4 h of incubation, the medium with MTT salt was removed and obtained crystals were dissolved in DMSO (dimethyl sulfoxide, 200 mL/well; POCH, Poland). The solution absorbency was measured at 570 nm using PowerWave™ microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). Each cytotoxicity assay was conducted three times and was measured in triplicates.

After 24 hours since incorporating BMSCs with a cell number of 5 × 10⁴ into the gel, the number of metabolically active cells within the hydrogels was measured using a MTT cell proliferation assay kit (Invitrogen). Cells plated on a glass with a number of 5 × 10⁴ were also examined as a control. The absorbance of samples treated with MTT reagents following the manufacturer's protocol was measured at 570 nm using a spectrophotometer (Synergy HT, BioTek). The neural differentiation was activated by incubating cells in a neurogenic differentiation media (PromoCell). After 7 days, following fixation and permeabilization, cells were incubated with the rabbit polyclonal anti-microtubule-associated protein 2 (anti-MAP2) (10 μg/mL, Invitrogen) or mouse monoclonal anti-glial fibrillary acidic protein antibody (anti-GFAP) (4 μg/mL, Sigma) overnight. Thereafter, cells were incubated with the secondary fluorescent antibodies to image intracellular MAP2 and GFAP using the laser-scanning confocal microscope (LSM700, Zeiss). Cells located at depth of 60 mm from the top surface of the gel were captured. Calcium channels of the differentiated BMSCs were imaged by staining cells with Fluo-4AM (Invitrogen). The positively stained calcium channel was imaged using the confocal microscope. Approximately 300 cells were imaged in 8 separate gel matrix.

The viability of the cells was examined by the standard MTT assay, using the MTT Cell Proliferation Assay kit (Invitrogen, US). In this test, the activity of mitochondrial enzyme-succinate dehydrogenase is used. This enzyme in living cells is responsible for the transformation of soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to water-insoluble purple formazan crystals. Following 4 h incubation, the medium with MTT was removed, and the formed crystals were dissolved in DMSO. The solution absorbency was measured at 540 nm, using the PowerWave™ microplate spectrophotometer (Bio-Tek Instruments, USA). The experiment was repeated 3 times, and the measurements were performed in triplicate.
The results were analysed statistically in the STATISTICA vs. 13 application (StaftSoft, Poland). Data were calculated as mean ± SD. To compare more than two groups, the one-way analysis of variance ANOVA and post hoc multiple comparisons on a basis of Tukey's HSD test were used. All parameters were considered statistically significantly different if p values were less than 0.05.

Viability of HRECs after treatment with Hcy (100, 50 and 20 µM) was determined using MTT Cell Proliferation Assay Kit (Invitrogen, Grand Island, NY, USA). HRECs were grown on 96-well plates at a density of 2 × 10⁴ cells/well in phenol red free medium and incubated with different concentrations of Hcy for 24 hours. The medium was removed and the cells were treated with 20 μL of MTT (5 mg/mL) and incubated for 4 h hours at 37 °C prior to the addition of 100 μL of DMSO. The resulting formazan product was measured spectrophotometrically at 540 nm using a microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA, USA).
Caspase-3/7 activity in homocysteine treated HRE cells was measured using the Apo-ONE Homogenous caspase-3/7 system according to the manufacturer’s instructions (Promega). The intensity of the emitted fluorescence was determined at an excitation wavelength range of 485 ± 20 nm and an emission wavelength range of 530 ± 25 nm using microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA, USA). Caspase-3/7 activity was expressed as net fluorescence intensity (RFU). Data shown are means ± SD of three different independent experiments.

For experiments, fibroblasts were harvested through trypsin-EDTA (Gibco) treatment, seeded into 12-well plates (Greiner bio-one, Germany) at a density of 10,000 cells/cm², and cultured for 24 h to 30% confluence. Afterwards, fibroblasts were incubated with the test specimen extracts (refer to sample preparation for details) for 24 to 72 h. Cells cultured in complete DMEM served as untreated control. Additionally, cells incubated with 10 ng/mL epidermal growth factor (EGF, Promocell) were used as positive control for proliferative effects. Determination of cell proliferation was carried out using the luminometric ATP assay (ATPLiteTM-M Assay, PerkinElmer). Here, the amount of viable cells was specified by cellular ATP-content in [nM]. The number of viable, metabolically active cells was further determined using the photometric MTT assay (MTT Cell Proliferation Assay Kit, Invitrogen). The cell amounts are given as the absorbance measured at 580 nm in [mOD].