Mtt solubilization solution

The viability of HUVEC and astrocytes was evaluated by 1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Life Technologies Italia, Monza, Italy) dye [15 (link)]. To do this, 50,000 HUVEC/astrocytes/well were plated in 24-Transwells plates in complete medium (DMEM supplemented with 10% FBS). HUVEC and astrocytes were treated with 10% plasma for 3 h, as described in the experimental protocol.
After each stimulation, the medium was replaced with fresh culture medium with 0% red phenol and 0% FBS. MTT dye was added to the well plates containing the cells and left in an incubator for 2 h at 37 °C. After that, the medium was replaced with a MTT solubilization solution (dimethyl sulfoxide; Sigma, Milan, Italy) and mixed until the complete dissolution of formazan crystals. The viability of HUVEC/astrocytes was evaluated by measuring the absorbance at 570 nm through a spectrometer (VICTOR™ X Multilabel Plate Reader). Control cells (not treated cells) were set as 100% viability. Experiments have been executed in triplicate and repeated three times on different pools of HUVEC and astrocytes.

Cytotoxicity was tested using a lactate dehydrogenase (LDH) release assay. An LDH cytotoxicity assay kit from BioVision (Mountain View, CA, USA) was used in the way recommended by the manufacturer. The optical density values were quantified by measuring absorbance at a wavelength of 450 nm using a microplate reader (EMax, Molecular Devices, Sunnyvale, CA, USA) [12 (link)].
Cell viability was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich, St. Louis, MO, USA) assay. This assay is based on the reduction of MTT by living cells to yield a soluble formazan product that can be colorimetrically detected. Briefly, MTT was added at 0.5 mg/mL in the culture medium of cells growing in 4-well plates. After a 2 h of incubation at 37 °C with 5% CO₂, the resulting formazan crystals were dissolved in MTT solubilization solution (Sigma–Aldrich) and quantified using an Emax spectrophotometer (Molecular Devices) by measuring the absorbance at 595 nm. The background absorbance at 690 nm was subtracted [12 (link)].

EK-1 cells were plated in 96-well plates at approximately 1.5 × 10⁴ cells per well. After 24 h, the medium was replaced with fresh medium containing different concentrations of gold nanoparticles as described above. Cellular viability of EK-1 cultures after introduction to the gold nanoparticles was examined as above. Any toxic effects were noted, such as cells becoming sub-confluent or altered in morphology compared with non-treated control cells. Plates were incubated at 26 °C for 1 week. The medium was then removed and the viability of cultures was assayed by incubating with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich), following the method described by Mosmann [38 (link)]: 20 μl MTT (5 mg/ml in balanced salt solution) was added to each well and incubated for 2 h. After that, medium of each well was discarded, and 200 μl of MTT solubilization solution (Sigma-Aldrich) was added and the plates agitated with an orbital shaker. Triplicate absorbance values of each well were measured in a microplate reader at 570 nm against a reference wavelength of 690 nm. The percentage of cell viability was calculated as the optical density values of treated cells divided by the mean optical density of non-treated cells, multiplied by100.

Monochloroacetic acid, chlorosulfonic acid, formamide, pyridine, trichloroacetic acid, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and MTT solubilization solution (10% Triton X-100 in acidic isopropanol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acyclovir (ACV, 10 μg mL⁻¹) was obtained from Novafarma Ind. Farmac. (Anápolis-GO, Brazil). All chemicals were of analytical grade.

SHSY5Y cells were plated on 96 well plates (Costar) at a density of 13000 cells per well of 100-μl of medium and were then differentiated. After the treatment periods, cell media were replaced with DMEM containing 10% 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) for 3 h at 37°C. Then, all remaining media and MTT solutions were collected, and the resultant formazan crystals were solubilized in 100 μl of MTT solubilization solution (Sigma) and shaken for 10 min. Absorbance was measured at 570 nm with an ELISA Reader (SynergyH4 Hybrid Reader, BioTek). Assay values obtained upon vehicle treatment were set as 100%, and complete inhibition of MTT reduction (0%) was defined as the value obtained following the addition of the MTT solubilization solution. Cell death was determined by the following formula: % of cell death = 100% − % of cell viability.

An in vitro toxicology assay kit (Sigma Aldrich) was used for this experiment. Briefly, NHLFs were plated into a 12-well plate and pretreated with ATO (10nM, 20nM) for 48 hrs. Cells were then washed with HBSS prior to addition of 1 ml of FGM. MTT (M-5655, Sigma Aldrich) was added into to media and cells were incubated at 37°C for 2 hrs. Thereafter, the culture media was removed and MTT Solubilization Solution (M-8910, Sigma Aldrich) was added. Absorbance of each well was measured at the wavelengths of 570 nm and 690 nm.