Lactate dehydrogenase ldh kit

In both in vitro and in vivo experiments, the rumen fluid was centrifuged at 10,000 × g for 10 min at 4°C and filtered through a 0.22 μm filter. A total of 0.2 mL of 20% metaphosphoric acid stock solution (Sinopharm Group, China) containing 60 mM crotonic acid (Sinopharm Group, China) was added to 1 mL of the supernatant for volatile fatty acid (VFA) determination. The VFAs were determined through capillary column gas chromatography (GC-14B, Shimadzu, Kyoto, Japan; film thickness of the capillary column, 30 m × 0.53 m × 1 μm; column temperature, 110°C; injector and detector temperature, 200°C) (9 ). The serum and rumen fluid LPS concentrations were measured using a Chromogenic End-point Tachypleus Amebocyte Lysate Assay Kit (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, China) as described previously (7 (link), 10 (link)). The lactate concentrations of rumen fluid and serum were measured using a lactate Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions protocol. The LDH release of the serum was detected using a lactate dehydrogenase (LDH) kit (Jiancheng Bioengineering Institute, Nanjing, China).

Notoginsenoside R1 (NGR1) (PubChem CID: 441934) was purchased from Tianjin Solomon Bio-technology Co., Ltd. (Tianjin, China). Metoprolol tartrate (Met) was bought from AstraZeneca Pharmaceutical Co., Ltd. (Shanghai, China). The primary antibodies against cyclooxygenase-2 (COX2) (Cat# ab179800), carnitine O-palmitoyltransferase 2 (CPT2) (Cat# ab110293), and platelet glycoprotein 4 (CD36) (Cat# ab252922) were obtained from Abcam (Cambridge, MA, United States). The primary antibodies against very long-chain specific acyl-CoA dehydrogenase (ACADVL) (Cat# PB1076) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat# BA2913) were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). The HRP-conjugated secondary antibodies (Cat# bs-40295G-HRP) were bought from Bioss, Inc. (Beijing, China). The lactate dehydrogenase (LDH) kit, creatine kinase-MB (CK-MB) kit, α-hydroxybutyrate dehydrogenase (α-HBDH) kit, free-fatty acid (FFA) kit, and enzyme-linked immunosorbent assay (ELISA) kits for rat interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), and tumor necrosis factor-alpha (TNF-α) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals and solvents applied were of analytical grade.

R-HPABA (optical purity > 99.3%) and S-HPABA (optical purity > 98.8%) were synthesized in School of Pharmacy, Shenyang Pharmaceutical University (Shenyang, China). Aspirin (purity ≥ 99.5%) was supplied by Shandong Xinhua Pharmaceutical Co., Ltd (Shandong, China). Adenosine diphosphate (ADP), collagen (COLL, Type I) and arachidonic acid (AA) were purchased from Shanghai Ryon Biological Technology Co., Ltd (Shanghai, China). Epinephrine hydrochloride injection was obtained from Grandpharma (China) Co., Ltd. Trisodium citrate and dimethyl sulfoxide (DMSO) were of analytical grade. Thromboxane B₂ (TXB₂) ELISA kit, 6-keto-prostaglandin F1α (6-keto-PGF1α) ELISA kit, cyclooxygenase-1(COX-1) ELISA kit and lactate dehydrogenase (LDH) kit were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. (China). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA).

Cell survival was determined using a cell counting kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan). mRTECs and HK-2 cells were seeded in 96-well plates at 3000 cells per well and treated with erastin or incubated under hypoxic conditions. Subsequently, 110 μl of fresh medium containing 10 μl CCK-8 solution was added to the cells, followed by incubation for 2 h (37 °C, 5% CO₂). Absorbance at 450 nm was then measured. LDH release was detected using a lactate dehydrogenase (LDH) kit (Nanjing Jiancheng Bioengineering Institute). mRTECs were seeded in 6-well plates at 1 × 10⁵ cells per well and treated with erastin or incubated under hypoxic conditions. The culture supernatant was collected and the cells were treated with 1.5% Triton X-100, and then the cells and supernatant were incubated with coenzyme I and 2,4-dinitrophenylhydrazine for 15 min at 37 °C. Absorbance was then determined at 490 nm.

Squid roes (S. oualaniensis) and sea cucumbers (Cucumaria frondosa) were provided by Tuandao (Qingdao, China). RPMI 1640 medium was provided by Gibco (New York, NY, USA). FBS was bought from Zeta Life (San Francisco, CA, USA). The lactate dehydrogenase (LDH) kit was donated by Jiancheng (Nanjing, China). GW9662 was donated by Sigma-Aldrich (St. Louis, MO, USA). The anti-PPARγ, anti-retinoid X receptor alpha (RXRα), anti-phosphatase and tensin homolog (PTEN), anti-phosphoinositide 3-kinase (PI3K), anti-serine-threonine protein kinase (AKT), anti-phosphorylated AKT(p-AKT), anti-C-X-C chemokine receptor type 4 (CXCR4), and anti-cofilin antibodies were provided by Abcam (Burlingame, CA, USA). The anti-Matrix metallopeptidase 9 (MMP9), anti-metallopeptidase inhibitor 1 (TIMP1), anti-TGF-β, and anti-cofilin antibodies were obtained from CST (Danvers, MA, USA). The anti-NF-κB and anti-phosphorylated NF-κB (p-NF-κB) antibodies were bought from Santa Cruz (Dallas, TX, USA). The anti-vascular endothelial growth factor (VEGF), anti-heparanase (Hpa), and anti-F-actin antibodies were acquired from Abcam (Cambridge, UK).

Zhilong Huoxue Tongyu capsule (Med-drug permit no. Chuan Z20070528; Patent number 200810147774.1) was provided by the Preparation Room for TCM at the Affiliated Hospital of TCM, Southwest Medical University. The specific information of antibodies (p-PI3K, PI3K, p-AKT, AKT, Nrf2, HO-1, GPX4, ACSL4, GAPDH) is presented in S1 Table. RNA extraction, reverse transcription, and reaction reagents were purchased from TOYOBO (Shanghai) Biotech Co., Ltd. The BCA protein concentration assay kit and protein lysis buffer were purchased from Beyotime Biotechnology Co., Ltd. The lactate dehydrogenase (LDH) kit, Creatine kinase-MB isoenzyme (CK-MB) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, reactive oxygen species (ROS) kit, and ELISA kit were purchased from Jiancheng (Nanjing) Biotech Co., Ltd.

After transfection, the cell fluid was collected and the lactic acid extraction kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China) was used to measure the content of lactic acid production during glycolysis process. In addition, the SCs were collected after transfection, and then the ATP kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China), lactate dehydrogenase (LDH) kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China), and pyruvate extraction kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China) were used to measure the change level of key enzymes, energy, and pyruvate content in the glycolysis process. Notably, all the experiments were conducted following the manufacturer’s instructions.