Cell proliferation kit 1 mtt

Cell Proliferation Kit I (MTT) (Sigma) was used to test cell proliferation consistent with the manufacturer’s instructions. Concisely, cells were adhered and grown in 96-well dishes with a density of 5×10³ cells/well overnight. The proliferation curves of cells, involving cultured cells in three days, were formulated by measuring absorbance at 570 nm.

To assess cell viability, we used the cell proliferation kit I (MTT) (Sigma-Aldrich, Castle Hill, NSW, Australia) following the procedures described in previous work [50 (link)]. Cells were seeded into 96-well plates at a concentration of 1 × 10⁴ cells/well. DMEM containing 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution was added in each well. Following incubation for 6 and 24 h at 37 °C, the medium was removed, and 100 μL of solubilization solution was added. Formazan formed by the cleavage of the yellow tetrazolium salt MTT was measured spectrophotometrically by absorbance change at 550–600 nm using a microplate reader.

Cell growth was evaluated by MTT assay (Cell Proliferation Kit I (MTT), Sigma-Aldrich, St. Louis, Missouri, MI, USA), according to the manufacturer’s instructions. Experiments were performed in triplicate. Apoptosis was evaluated by flow cytometry measuring annexin staining. Briefly, cells were washed once with PBS 1× and incubated for 15 min with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (Annexin V-FITC Apoptosis Detection Kit, Immunostep, Salamanca, Spain). After incubation, cells were analyzed by flow cytometry. For all samples, at least 100,000 events were acquired. BD CellQuest software (BD Biosciences, New Jersey, NJ, USA) was used for data analysis.

The ability of a peptide to cause a harmful or toxic effect on human cells is referred to as its cytotoxicity activity [48 (link)] A cytotoxicity assay was performed as described by Shwaiki, Arendt and Lynch, 2020 using a colonic cell line [44 (link)]. Briefly, Caco-2 cells (ECACC) were prepared in Dulbecco’s modified Eagle media (DMEM) that had been supplemented with 1% nonessential amino acids and 10% foetal bovine serum (FBS). A cell suspension of 1 × 10⁵ cells/mL was prepared, 200 µL of which was added into the wells of a flat-bottom 96 well microtitre plate and incubated for 24 h at 37 °C with 5% CO₂. Following this incubation, the media were removed and D-lp1 was added into the wells at concentrations ranging from 100 to 700 μg/mL, together with DMEM supplemented with 2.5% FBS. Each well contained a final volume of 200 μL. The controls consisted of sterile water in DMEM. The plate was incubated at 37 °C for 24 h. The media in the wells was discarded and 100 μL of DMEM and 10 μL of the MTT labelling reagents (Cell proliferation Kit I MTT; Sigma, Ireland) were transferred to each well. Following a 4 h incubation period, a solubilisation buffer (100 μL) was added, and the plate was incubated for a further 24 h. A fluorometric spectrophotometer was used to measure the cell viability in each well at a wavelength of 570 nm using a background reading of 690 nm.

Phosphate buffered saline (PBS), ethylenedyaminetetracetic sodium salt (EDTA), HEPES, bovine serum albumin (BSA), RPMI 1640 and Hank's balanced salt solution (HBSS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 6-propyl-2-thiouracil (PTU) and 2-ethylhexyl-4-methoxycinnamate (Octyl Methoxycinnamate—OMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IgG anti-murine CD3 (clone 145-2C11), APC-conjugated hamster IgG anti-murine CD3, PE-conjugated hamster IgG anti-murine CD8, and FITC-conjugated rat IgG anti-murine CD4 were all obtained from EXBIO Praha (Vestec, Czech Republic). Fetal bovine serum was obtained from Hyclone (Logan, UT, USA). Carboxyfluorescein diacetate-succinimidyl ester (CFSE) was obtained from Invitrogen (Carlsbad, CA, USA). Cell Proliferation Kit I (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Thyroxine (T4) AccuBind® kit was purchased from Monobind Inc. (Lake Forest, CA, USA).

The splenocyte proliferation was measured by the Cell Proliferation Kit I (MTT) of Sigma-Aldrich (St. Louis, MO, USA) according to the manufacturer's protocol. Splenocytes, recovered from 3 week old male Swiss mice, were treated for 1 h with different concentrations of OMC (1–200 μg/mL) or vehicle; and subsequently cultured in the anti-CD3 mAb-coated wells at a concentration of 10⁵ cells/well in RPMI 1640 medium supplemented with 10% FBS (at 5% CO₂ and 37°C). After 72 h, the cells were incubated with the MTT solution for another 4 h. The water insoluble formazan dye was solubilized before the measurement of absorbance using a Spectramax M5 multiwell spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The absorbance was read at 550 nm (25 (link)).