Cell viability was assessed by colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay, as described by Mosmann [23] (link), with modifications. The cells used were murine macrophages obtained by washing the peritoneal cavity from Swiss mice and Cell Line RAW 264.7. The compounds to be tested were diluted with 0.1% DMSO in culture medium in serial dilution to obtain seven concentrations ranging 1000–15.6 μg/mL (1:2; 7× dilution), corresponding to 3773–58.9 μM for the 4a derivative; 3390–53.0 μM for derivative 4c and 3413–53.3 μM for 4d . For tests, RAW 264.7 line cells (1 × 104 cells per well) and macrophages from primary culture cells (1 × 106 cells per well) were distributed into 96 well microplates. After 48 h incubation at 37 °C, 20 μl of MTT solution (5 mg/mL) were added to each well and, after 3 h, the supernatant was removed and 200 μl of DMSO were added. The optical densities of the plates were read by a microplate spectrophotometer (Thermo Plate ®) at 570 nm. The IC50 for the primary cell culture and RAW cells was determined using GraphPad PRISM software ®. The test was performed in three repeats.
Thermo plate
Manufactured by Thermo Fisher Scientific
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The Thermo Plate® is a laboratory equipment designed for heating and incubating samples. It provides a controlled temperature environment for various applications in life science and analytical laboratories.
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2 protocols using thermo plate
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Colorimetric MTT Cell Viability Assay
2
Cytotoxicity Assessment by MTT Assay
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The cytotoxicity of the tested formulations was measured by the metabolizing ability of mitochondrial dehydrogenases to convert MTT into formazan [45 (link)]. After treatments, cells were incubated with 100 µL of medium with MTT (1 g/L) for 2 h. After a period of incubation of 2 h, cells were lysed and formazan crystals were dissolved with 100 µL of 20% (w/v) sodium dodecyl sulfate in a solution of 50% (v/v) N,N-dimethyl formamide in reagent grade water at pH 4.7 and left overnight at 37 °C [46 (link)]. The absorbance was measured at 595 nm using a microplate reader (THERMO PLATE, Thermo Fisher Scientific, Waltham, Massachusetts, United States). The results were expressed relative to the control group, which was considered as having 100% viability. The results are shown as cell viability, which corresponds to the mitochondrial dehydrogenase activity. All cultures were also examined by phase contrast microscopy with a green filter (LabomedR TCM400 Microscope, Tokyo, Japan) to evaluate morphological alterations and cellularity.
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