3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium

SH-SY5Y (ATCC® CRL-2266TM) cell line was purchased from ATCC (American Type Culture Collection, USA). (R,S)-salsolinol (SAL) of purity ≥ 99% was obtained from Cayman Chemical (USA). MPP⁺ (1-methyl-4-phenylpyridinium iodide) of purity ≥ 98% was purchased from Angene (India). The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay with a novel tetrazolium compound, namely 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, USA). Rhodamine 123 and Hoechst 33,258 were purchased fro Sigma-Aldrich (Merck LifeScience, Poland) and ThermoFisher Scientific (USA), respectively.

Chitosan (50 kDa, deacetylation degree 85%) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd, Shandong, China. Insulin (INS, 21 IU/mg) was from Dingguo Biotech Co., Ltd, Shanghai, China. HPMCP (HP-55S) was from Huzhou Mizuda Hope Bioscience Co., Ltd, Huzhou, China. Alloxan was from Sigma-Aldrich (Chicago, IL). Fluorescein isothiocyanate (FITC) and Rhodamine B (RhB) were from Tokyo Chemical Industry Co., Ltd, Tokyo, Japan. Sulfo-Cyanine5 NHS ester (Cy5) was from Lumiprobe, Hunt Valley, MD. Bicinchoninic acid (BCA) protein assay kit was from Thermo Fisher Scientific Inc, Waltham, MA. DMEM, fetal bovine serum, MEM non-essential amino acid solution, L-glutamine, penicillin and streptomycin were from GIBCO BRL Life Technologies Inc., Carlsbad, CA. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was from Promega Co., Madison, WI. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was from Beyotime Institute of Biotechnology, Jiangsu, China. DAPI Fluoromount-G^TM was from Yeasen Biotechnology, Shanghai, China. All other chemicals were of analytical grade and from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China.

Cells were seeded in 96-well cell culture plates and incubated overnight before serum starvation for 24 hours. Cells were then treated with hrGH (1.0 µg/mL) with or without pegvisomant. To measure the viability of the cells, 20 µL of the MTS reagent 3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Promega, Madison, WI) was added to each well and cells were incubated for 1 hour at 37°C in 5% CO₂. Absorbance at 490 nm was detected using a plate reader (CLARIOstar; BMG Labtech, Cary, NC).

The human monocyte THP-1 cell line (ATCC TIB-202) was used to evaluate the toxicity levels of metallic- and ZnO-NPs as well as the combination of both NPs. THP-1 cells were grown as monocytes in RPMI medium supplemented with heat deactivated 10% fetal calf serum (Hyclone, GE, IL, USA), and a mixture of penicillin (100 μg/mL) and streptomycin (100 U/mL) (StemCell, Vancouver, BC, Canada). THP-1 cells were incubated at 37°C in a humid atmosphere of 5% CO₂. Cells (1x10⁴ cells/well) were activated the night before of the experiment by the addition of 40 ng/mL phorbol myristate acetate (PMA, Sigma). The next day, the medium was aspirated and replaced with fresh media without antibiotics and exposed to the NPs for 24 h using the same incubation temperature as stated above. Thereafter, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, WI, USA) was added to the wells and the optical density of the plates was read at 450 nm using a plate reader (Epoch, Tecan, Männedorf, Switzerland). Staurosporine (1 μg/mL, Sigma) and untreated cells were used as positive and negative controls, respectively. The cytotoxic activities were performed without exposure to the light to mimic the environment once the NPs are penetrating into the body.

For the MTS assay, HEK Blue cells were cultured and prepared in the same manner as in the CD40 sensor assay. Then cells were added to a 96-well microtiter plate at a density of 50,000 cells/well in the absence or presence of various concentrations of compounds diluted in the same media, without the addition of CD154. The plate was incubated at 37 °C for 24 h. 20 µL per well of MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, (Promega, Madison, WI, USA) was added to the culture after treatments, and cells were incubated at 37 °C for 4 h. Formazan levels were measured using a plate reader at 490 nm.