Resazurin sodium salt

In our study, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS solution), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N,N′,N′-tetramethylethylenediamine (TEMED), acrylamide/bisacrylamide, ammonium persulfate (APS), ascorbic acid (vitamin C), bovine serum albumin (BSA), ethanol (96%), glycine, methanol, Ponceau S, resazurin sodium salt (RES), sodium dodecyl sulfate (SDS), Trolox, trypsin-EDTA solution, Tris-HCl, Tris-Base, and Tween-20 were purchased from Merck KGaA (Darmstadt, Germany). The PVDF membrane with 0.45 µm-pore size and primary antibodies against SOD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies against GAPDH, NF-κB, IκBα, SRC, GLUT4, PCNA, PGC-1α, PPARγ, p-ERK1/2, and ERK1/2 were purchased from ABClonal (Woburn, MA, USA). The primary antibodies against NLRP3, anti-mouse- and anti-rabbit-HRP-conjugated antibodies were purchased from ThermoFisher (Waltham, MA, USA). Potassium persulfate (Warchem, Zakręt, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), DMEM (Dulbecco’s Modification of Eagle’s Medium, Biological Industries, Beit Haemek, Israel), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), and phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Ślaskie, Poland) were also used during the analyses.

The commercial product Ecotrich WP 1 × 10¹⁰ UFC/g (Balagro^™, Brazil) was used to obtain the initial culture of T. harzianum to synthesize the nanoparticles. Titanium IV Rutile oxide (≥ 99.98% purity), MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) salt (≥ 97.5% purity), Trypan Blue dye and Resazurin sodium salt were purchased from Sigma Aldrich Chemicals, USA. Potato dextrose agar (PDA), potato dextrose broth (PDB) and Müeller Hinton broth (MHB) were obtained from Kasvi, Brazil. Dimethylsulfoxide (DMSO) (99.9% purity) was obtained from Dinâmica, Brazil; Power Soil^™ DNA Isolation Kit was purchased from Qiagen. Qubit^™ RNA HS Assay Kit and SUPERSCRIPT^™ III RT were purchased from Thermo Fisher Scientific, USA. Other reagents were acquired analytically from local suppliers (Sorocaba, Brazil). Cytotoxicity and genotoxicity evaluations were performed using three cell lines (Table 1) obtained from BCRJ (Cell bank of Rio de Janeiro).Table 1

Cell lines used in cytotoxicity and genotoxicity assays: species, origin/code and technical characteristics

Cell line	Species	Origin	Technical characteristics
HaCat	Homo sapiens	BCRJ Code 0341	Tissue: Skin Cell type: keratinocytes
V79-4	Cricetulus griséus (Chinese Hamster)	BCRJ Code 0244	Tissue: Lung Cell type: Fibroblasts
3T3—Swiss albino	Mus musculus (Swiss albino)	BCRJ Code 0017	Tissue: Embrionary Cell type: Fibroblasts

The tolerance of human corneal fibroblasts to C₁₆-YEALRVANEVTLN was determined using the Alamar blue assay, which correlates cellular metabolism to the reduction of a resazurin sodium salt (Sigma-Aldrich, Dorset, UK) by mitochondrial dehydrogenase activity. Fibroblastic cells, 1 × 10⁴ per well, were seeded (serum free) into 24 well plates (Corning) and allowed to adhere overnight before exposure to 0.00125 wt% and 0.0025 wt% solutions of PA (either in monomeric or aggregated form prior to dilution) in SFM. Cultures were maintained for seven days or three weeks. PA-supplemented and un-supplemented (control) growth media were replaced every two days over the culture periods. At the end of these culture periods media were replaced with resazurin reagent (1 : 10 in SFM) and incubated for 3 hours (37 °C/5% CO₂) at which point 100 μl samples were taken in duplicate from each well and assayed for fluorescence emission at 590 nm under excitation at 544 nm using a Fluoroskan Ascent™ (Thermo scientific, Paisley, UK). The cell number was calculated by interpolation using a standard curve for the fluorescence values derived from known numbers of cells, determined in parallel with each separate assay. The PA solutions had no effect, in either monomeric or diluted aggregate form, upon the reduction of resazurin in the absence of cells.

Cytotoxicity of the MTAbl peptides towards HeLa cells was evaluated before the cell internalization assay. The mammalian cell cytotoxicity assay was conducted using a method described earlier65 (link). HeLa cells were seeded the day before the assay in 96-well tissue culture-treated plates (2.5 × 10³ cells/well). Peptides TAT, MCoTI-II, MTAbl06, 07, 09, 13 and 15 were dissolved and diluted in sterile H₂O (2-fold dilutions starting from 640 μM). Peptide solutions were diluted 10-fold with serum-free medium and incubated with cells in triplicates for 2 h. Controls with H₂O or 0.01% (v/v) Triton X were included to have 100% and 0% cell viability, respectively. Following incubation the peptide solutions were removed and aliquots of 10 μL of 0.02% (w/v) sterile resazurin (resazurin sodium salt, Sigma) were added to each well containing 100 μL of fresh medium. Cells were incubated under standard conditions of 37 °C and 5% CO₂ for 22 h. The absorbance of the plates was measured on a plate reader at 540 and 620 nm. The cytotoxicity of peptides abltide, MCoTI-II, MTAbl06, 07, 13 and 15 against K562 cells was evaluated using the resazurin-based assay with 5 × 10³ cells per well.