Enzyme labeling instrument

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).

CCK-8 (ck04, Dojindo, China) was used to evaluate cell proliferation. A 100-μl cell suspension with a density of 1 × 10⁴ cells/ml was added to each well of a 96-well plate, and triplicate wells were established for each group. Then the plate was placed in an incubator (37 °C, 5% CO₂) overnight. At 24, 48, 72, and 96 h, 100 μl of medium containing 10% CCK-8 reagent was added to each well. After incubation for 2 h in an incubator, the optical density (OD) value at 450 nm was measured in an enzyme labeling instrument (BioTek, USA). A cell growth curve was drawn with time as the abscissa and the OD value as the ordinate. The experiment was repeated three times.

H2050r high-speed refrigerated centrifuge (Hunan Xiangyi Company), MIX-S vortex mixer, shaker oscillator (Shiloh, USA), TGear mini centrifuge (Tiangen), heating magnetic agitator (Dalong, Beijing), SIM-F140 ice maker (Sanyo, Japan), electronic balance (Sartorius, Germany), enzyme labeling instrument (BioTek, USA), tissue grinding homogenizer (MP Biomedicals, USA), electric constant temperature blast drying oven (Jinghong, Shanghai), electrophoretic system, transfer system, glue rack, ultralow-temperature freezer (SANYO, Japan), microtome (Laika, Germany), and TKY-BMB, electrothermostatic water bath (Hualida).

A 2 mL MAC-T cells suspension (5 × 10⁴ cells/mL) was seeded into the wells of a 6-well plate. After the cell confluence reached 70–80%, the completed medium was removed, and the cells were washed twice with cold PBS. Then, the cells were incubated with treated medium. The MDA content of the MAC-T cells was determined using a microplate method kit (Cat No. A003-4-1; Nanjing Jiancheng Biotechnology Institute, Nanjing, China). The total oxidant status (TOS, Cat No. BPE92369), total antioxidant status (TAS, Cat No. BPE92207), TNF-α (Cat No. BPE92091), IL-1β (Cat No. BPE92157), IL-6 (Cat No. BPE92153), and IL-10 (Cat No. BPE92159) of the MAC-T cells were detected using ELISA kits (Shanghai Lengton Bioscience Co., Ltd., Shanghai, China) following the manufacturer's instructions. Briefly, MAC-T cells were digested by trypsin and placed in centrifuge tubes. Then, the tubes were centrifuged at 2,000 rpm for 20 min, and the supernatant was collected. After the cells were counted, the cell suspension was adjusted with PBS to reach a cell concentration of 1 × 10⁶ cells/mL. Absorbance was detected at 530 (MDA) and 450 nm (TOS, TAS, TNF-α, IL-1β, IL-6, and IL-10) using an enzyme-labeling instrument (BioTek, Hong Kong, China).

HCC1806 cells were (5 × 10³ cells/well) seeded into 96-well plates overnight and then treated with GA (0, 200, 250, and 300 μM) for 24, 48, and 72 h. The MTT (5 mg/ml) reagent was added to each well (20 μL) and cultured for another 4 h. Next, dimethyl sulfoxide (DMSO) was added to each well (150 μL), and the absorbance was recorded at 490 nm by a Enzyme labeling instrument (BioTek, United States). MDA-MB-468 cells were (5 × 10³ cells/well) seeded into 96-well plates overnight and then treated with GA (0, 3, 6, 12, 24, 48, 96, 192, 288, and 384 μM) for 24 h. Other steps are the same as those of HCC1806 cells (Yu et al., 2016 (link)).

BRL cells were transfected with RNA oligonucleotide or plasmid DNA. Fore hours after transfection, equal numbers of viable cells were seeded in 96-well plates for cell proliferation assay. Cell growth was determined using CCK8 kit. In brief, one-tenth volume of CCK-8 was added to each well, and the cells were cultured for another 2 h. Cell density was determined by measuring the absorbance at 450 nm using an enzyme-labeling instrument (BioTek, Winooski, VT, USA).

An enzyme labeling instrument (Bio Tek Instruments, Inc., Vermont, VT, USA) was used to determine total cholesterol (T-CHO cat. NO. F002-1-1), triglyceride (TG cat. NO. A110-1-1), high-density lipoprotein (HDL cat. NO. A112-1-1), low-density lipoprotein (LDL cat. NO. A113-1-1), total blood glucose (GLU cat. NO. YX-071221C), growth hormone (GH cat. NO. YX-070800C) and insulin (INS cat. NO. YX-091419C) in plasma. All kits were purchased at Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) and determined strictly according to the instructions.