N acetyl l cysteine

Sputum samples digested and decontaminated by the N-acetyl l-cysteine (NALC) (Sigma–Aldrich, Germany) and 3% sodium hydroxide (NaOH) (VWR, Belgium) method were processed for mycobacterial culture using the Bactec™ MGIT™ 960 system (Becton Dickinson, USA) as previously described [24] (link). Days to positivity (DTP), defined as the time it took for MGIT cultures to become positive were used as an inverse measure of the bacillary load in the sputum sample. Smears were prepared from positive MGIT cultures, stained with ZN stain and examined for AFB by light microscopy. The presence of Mtb in all cultures that were positive for AFBs was confirmed using the MPT64 antigen test (Becton Dickinson, USA) according to the manufacturer's instructions. MGIT cultures were positive for Mtb if both smear microscopy for AFB and MPT64 antigen test results were positive. They were reported negative if there was no growth after 42 days incubation.

Hyflo Super Cel (Diatomaceous earth), 3-aminopropyl(diethoxy)methylsilane (APDMS, 97%), dimethyl suberimidate dihydrochloride (DMS, 98%), lysozyme solution (50 mg/mL in distilled water), sodium hydroxide solution (50% in H₂O), N-Acetyl-L-cysteine (NALC, 99%), sodium citrate, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCI (pH 8.0), distilled water (DNase/RNase-Free), and EDTA (pH 8.0) were purchased from Invitrogen (Waltham, MA, USA). Proteinase K solution (>600 mAU/mL) was purchased from Qiagen (Hilden, Germany). Absolute ethanol was purchased from Merck (Whitehouse Station, NJ, USA). Phosphate-buffered saline (PBS; 10×, pH 7.4) was purchased from Gibco (Grand Island, NY, USA).

Cells were seeded in 6-well plates at a density of 2 × 10⁶ cells per well, and cultures were passaged by dissociation in 0.05% (w/v) trypsin in phosphate-buffered saline (PBS) pH 7.4 upon the confluence. For the pretreatment studies, the cells were treated with HBA (IUPAC name: 4-(Hydroxymethyl)phenol; Sigma Chemical Co., St. Louis, MO, USA; Cat. # H20806, purity ≥ 99%) at indicated concentrations (40, 80, 120, 160 µmol/L), 1 mmol/L N-acetyl-L-cysteine (NALC; Sigma Chemical Co., St. Louis, MO, USA; Cat. # A9165) or 10 µmol/L SP600125 (Sigma Chemical Co., St. Louis, MO, USA; Cat. # 420119) for 1 h, followed by exposure to a range of concentrations (25, 50, 100, 150, and 200 µmol/L) of 6-OHDA (IUPAC name:5-(2-aminoethyl)benzene-1,2,4-triol; Sigma-Aldrich, St. Louis, MO, USA; Cat. # H4381) for 24 h with no medium change. The powders of the experimental compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA; Cat. # D8418) to form a 1 mmol/L stock solution and then diluted in a culture medium at concentrations appropriate for later experiments. The final concentration of DMSO was below 0.1% (v/v), which is not usually an observable toxic effect for most cells [22 (link)]. Each condition was tested with three cell wells for each replicate, and each experiment was carried out a minimum of five separate times

N-acetyl-L-cysteine (NALC) and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The NaOH (0.4%) and NALC (0.025%, 1%, 2% and 4%) were prepared in distilled water. A phosphate buffer saline solution (0.01 M PBS) was prepared using standard protocols. Carbol fuchsin, 0.3%, was prepared by dissolving 50 g of phenol in 100 mL 90% ethanol and adding 3 g of basic fuchsin in the mixture which was brought to 1 L by adding distilled water. The decolorization solution was 25% sulphuric acid. The counter stain was 0.3% methylene blue. GMNPs were provided by the Alocilja Research Group from Michigan State University (USA). Briefly, GMNP (100 ± 58 nm) has a magnetite (Fe₃O₄) core and a glycan (chitosan) coating. Fe₃O₄ was synthesized using ferric chloride hexahydrate (FeCl₃·6H₂O) as a precursor in a mixture of ethylene glycol (as a reducing agent) and sodium acetate (as a porogen). Chitosan was polymerized to surface-modify the iron oxide nanoparticles.

The free and conjugated phenolic acids tested in this study are listed in Figure 2; 3,4-dihydroxyphenylacetic acid (DHPA) and 3,4-dihydroxybenzoic acid (protocatechuic acid; PCA) were purchased from Sigma-Aldrich (San Louis, MO, USA). Dihydrocaffeic acid (3-(3′,4′-dihydroxyphenyl)propionic acid, DHCFA) and its conjugated forms [dihydrocaffeic acid 3-O-β-D-glucuronide (DHCFAg) and dihydrocaffeic acid 3-O-sulfate (DHCFAs)] were purchased from Toronto Research Chemicals, Inc. (TRC, Toronto, QC, Canada). N-acetyl-L-cysteine (NALC), which is a common antioxidant that can directly react with ROS, promote glutathione synthesis, and inhibit nerve cell apoptosis, was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and was used as the antioxidant reference control [31 (link)]. Stock standard solutions of these compounds (1 mM) were freshly prepared and filtered (0.22 μm) in sterile Dulbecco’s phosphate buffered saline solution (DPBS, Lonza, Walkersville, MD, USA).
The bacterial lipopolysaccharide (LPS), a pro-inflammatory inducer, and tert-butyl hydroperoxide (tBHP), used to induce in vitro cellular oxidative stress, were purchased from Sigma-Aldrich (San Louis, MO, USA).

In this experiment, we used human MSCs from three healthy donors. Human ADSCs from two donors were tested for chondrogenic and adipogenic differentiation and one of ADSCs was used as a tool for EV production; WJ-MSCs (RM60596) were purchased from the Bioresource Collection and Research Centre (BCRC), Hsinchu, Taiwan, and were also used as an EV production tool. All cells were provided by Gwo Xi Stem Cell Applied Technology Co., Ltd. (Taipei City, Taiwan) and donors signed a consent form for cell donation.
The cells were cultured and maintained in keratinocyte serum-free medium (KSFM; Invitrogen-Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT, USA), antioxidants N-acetyl-L-cysteine (Sigma-Aldrich, St. Louis, MO, USA), and L-ascorbic acid 2-phosphate (Sigma-Aldrich). The cells were incubated at 37 °C with 5% CO₂ until the monolayer of adherent cells reached 70–80% confluence. During the experiment, the serum-free medium (SFM) was keratinocyte serum-free medium containing N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate, which was used as a production medium for collecting EVs. Exosome-depleted FBS (EV-depleted FBS) was purchased from Gibco (Invitrogen-Gibco). The cells were used for the study in the passages 5.