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Nanomaterials 2020, 10, 422 5 of 16 homogenate was centrifuged at 20,217× g for 1 h at 4 ◦C. The protein concentration was evaluated using the Lowry method [28] and the antioxidant enzyme activities were determined as follows. Glutathione S-transferase (GST) activity was measured spectrophotometrically through determining the glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) conjugates at 340 nm according to the method of Chitme et al. [29]. Catalase (CAT) activity was evaluated by quantifying the decrease in absorbance of hydrogen peroxide at 240 nm according to the method of Kumar et al. [30]. Superoxide dismutase (SOD) activity was measured using the adrenochrome test, which is dependent on the capability of SOD to suppress the autoxidation of epinephrine in alkaline conditions using the method of Lhinhatrakool and Sutthivaiyakit [31]. Glutathione reductase (GSR) activity was evaluated following the method described by [32], where one unit of glutathione reductase activity is known as the amount of the enzyme catalyzing the reduction of 1 μM of NADPH per min. Glucose 6 phosphate dehydrogenase (G6-P) activity was measured by quantifying the reduction of NADP at 340 nm following the previous method of Gupta and Sanjrani [33]. The activity of glutathione peroxidase (GPX) was determined by measuring a decrease in absorbance at 340 nm, suggestive of the disappearance of NADPH as described [32]. The reaction was initiated by the addition of hydrogen peroxide and the enzyme activity was calculated as nanomoles of NADPH oxidized per minute per milligram protein by using a molar extinction coefficient of 6.22 × 103 mol L−1 cm−1. 2.10. Transmission Electron Microscopy The aliquots from non-treated cells (control) and treated cells were washed, fixed in a solution of 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Sigma), and then washed in 0.1 M cacodylate buffer at pH 7.2. The samples were then post-fixed in 2.0% osmium tetroxide in 0.1 M cacodylate buffer at pH 7. The samples were dehydrated in a series of alcohol and placed in acetone. Infiltration and embedding were done in Epon-812 resin (EMBed-812 Embedding Kit, catalog no. 14120, Electron Microscopy Sciences, Hatfield, PA, USA). Sections were cut with a Porter Blum MT-2 ultra-microtome (Sorval, Liverpool, NY, USA) using glass and diamond blades. Ultrathin sections were compared in a solution of 2.0% uranyl acetate and lead nitrate/acetate. Samples was examined under a JEOL 1200EX II transmission electron microscope (Peabody, MA, USA) [34]. 2.11. Cell Culture Primary mouse BMSCs were isolated from sacrificed 8-week-old male C57BL/6J mice, as previously described [35]. In brief, bone marrow was flushed out from mouse tibia and femurs using BPS and collected in Eppendorf tubes. Bone marrow cells were isolated using centrifugation for 1 min at 400× g. Cells were purified via filtration through a 70-μm nylon mesh filter and cultured in 60 cm2 flasks in RPMI-1640 medium supplemented with 12% FBS (Gibco Invitrogen, Dreieich, Germany), 1% penicillin/streptomycin (P/S) (Gibco Invitrogen), and 12 μM L-glutamine (Gibco Invitrogen) in a 5% CO2 incubator at 37 ◦C. The non-adherent cells were removed after 6 h and cultured in fresh medium. The medium was changed every 3–4 days and BMSCs were maintained to be subcultured at a split ratio of 1:2. A human breast adenocarcinoma MCF-7 cell line was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (ACC 115, Braunschweig, Germany) [36]. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 10 μg/mL insulin (Gibco Invitrogen). 2.12. Cytotoxicity Assay The cytotoxicity of different compounds was determined by measuring the cell viability using an MTT cell proliferation assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. The cells were seeded in 96-well plates and treated with different concentrations of either AgNPs or AgNPs/CG for 48 h. The cells were then washed with PBS and incubated with fresh medium containing 0.5 mg/mLPDF Image | Inhibition of Candidiasis Calotropis Silver Nanoparticles
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