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18 protocols using sod kit

1

Brain Tissue Homogenization and Oxidative Stress Assessment

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About 200 ± 50 mg brain tissues of prefrontal cortex (PFC) was taken and washed by precooled normal saline (NS) for at least three times. And then, they were converted to 100g/L of brain homogenates in a homogenizer filled with nine times the mass of precooled NS. The homogenates were centrifuged at 4°C for 20 min at a speed of 3500 r/min. The protein quantification of supernatant was estimated by BCA method. And then, proper amount (50–100 μg) of supernatant’s lipid peroxidation levels (MDA) and SOD activity were measured according to the specifications of MDA kit (S0131, Beyotime, China) and SOD kit (S0101, Beyotime, China).
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2

Oxidative Stress Levels Assessment

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For the intracellular or serum oxidative stress levels, SOD kit (Beyotime, S0103, China) and MDA kit (Beyotime, S0131S, China) were applied. The total proteins of PDLCs or serum were immediately detected by using SOD or MDA kit following to the manufacturer's instructions and detected by a microplate reader (Thermo Fisher Scientific, USA). The assays were repeated three times.
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3

Hippocampal SOD Activity Measurement

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The activities of SOD in the hippocampus were measured using a SOD kit (S0101M, Beyotime, China) according to the manufacturer's instructions [32 (link)]. Add the SOD sample preparation solution provided by this kit according to the protocol for perfusion of brain samples. Use the BCA method to measure the sample concentration before adding the WST-8/enzyme working solution. One unit of SOD activity was defined as the amount that reduced the absorbance at 450 nm by 50%.
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4

Oxidative Stress and Cell Viability Assays

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UDCA (MCE, cat: HY-13771); fetal bovine serum (Biological Industries); trypsin (Beyotime, cat: C0201), penicillin–streptomycin (Beyotime, cat: C0222), reactive oxygen fluorescent probe (DCFH-DA, Yeasen, cat: 50101ES01); reverse transcription kit (Vazyme; code: R333-01); DMEM medium (Gibco), hydrogen peroxide (H2O2, Sigma, CAS-No: 7722-84-1); SYBR Green Mix (Vazyme); CCK-8 kit (Beyotime, cat: C0037); H2O2 kit (Solarbio, cat: BC353); and H2O2 kit (Solarbio, cat: BC3595); MDA kit (Beyotime; cat: S0131S); SOD kit (Beyotime; cat: S0101S); ROS kit (Yeasen; cat: 50101ES01).
Enzyme Labeling Instrument (BioTek, cat: Cytation3); Fluorescence Quantitative PCR Instrument (Applied Biosystems, USA, ABI PRISM®7900HT system).
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5

Antioxidant Activity Analysis of H9c2 Cells

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H9c2 cells were seeded in six-well plates at a density of 4 × 105 cells/well. Following treatment of Dox or co-treatment with Dox plus LAP, the activity of SOD, CAT and GSH in cell media was analyzed with a SOD kit from Beyotime Institute of Biotechnology (Jiangsu, China). The absorbance at 532 nm was recorded using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc.) [17 ].
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6

Quantifying Oxidative Stress Biomarkers

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Pancreatic tissue homogenate was prepared to detect the contents of malondialdehyde (MDA), 8‐OHdG, and superoxide dismutase (SOD) using MDA kit (S0131S, Beyotime), 8‐OHdG kit (ab201734, Abcam), and SOD kit (S0086, Beyotime) based on instructions of manufacturers.
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7

Assessing Oxidative Stress in BSMCs

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The MDA and SOD levels in BSMCs were detected by MDA kit (Beyotime Biotechnology, China) and SOD kit (Beyotime Biotechnology, China) respectively according to the manufacturer’s instructions.
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8

Quantifying Cellular Antioxidant Levels

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After treatment for 24 h, the total cellular protein of each group was lysed using RIPA buffer and quantified using a BCA protein assay kit (Beyotime) for detection of the SOD and GSH levels in the supernatant using a SOD kit (Beyotime) and a GSH assay kit (Beyotime), respectively. The data were obtained using a SpectraMax Plus384 spectrophotometer (Molecular Device, Sunnyvale, CA, USA), according to the manufacturer’s instructions.
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9

Neuroprotective Effects of Endophytic Extracts

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Dendrobine (purity ≥98%) was obtained from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Two endophytic strains were isolated from Dendrobium in our previous studies and named as Pseudomonas protegens CM-YJ44 (National Center for Biotechnology Information NCBI accession No.MZ674076), Priestia megaterium D-HT207 (NCBI accession No.MK389456), respectively. The dried extract of CM-YJ44 and D-HT207 was collected following the method by Wang et al. [13 ]. The extracts were redissolved in 1 mL Dimethyl Sulfoxide (DMSO) for the following experiments.
The human neuroblastoma SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). Dulbecco's modified Eagle's minimum (DMEM) was purchased from Thermo Scientific; fetal bovine serum (FBS) from Gibco; cell Counting Kit-8 (CCK-8) test, ROS kit, MDA kit, GSH kit, CAT kit from Solarbio; the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) fluorescent probe, SOD kit, NO kit and LDH kit from Beyotime; annexin V-FITC Apoptosis Detection Kit from BD; prime Script RT reagent Kit, SYBR Premix Ex Taq from TaKaRa.
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10

Oxidative Stress and Neurotransmitter Assays

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Contents of acetylcholine (ACh), malondialdehyde (MDA), activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and acetylcholinesterase (AChE) were measured using assay kits (ACh, MDA, LDH, and AChE kits: Nanjing Jiancheng Company, China; SOD kit: Beyotime Institute of Biotechnology, China). The absorbance was detected using an EnSpire microplate reader (PerkinElmer, United States).
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