High density lipoprotein cholesterol assay kit

The antibodies against MiD51, Drp1, p-Drp1, ERK1/2, p-ERK1/2, p27^Kip1, Cyclin D1, VCAM-1, ICAM-1, HIF-1α and CD31 were purchased from Proteintech (Wuhan, China). Bovine Serum Albumin (BSA) and 4, 6-diamidino-2-phenylindole (DAPI) were obtained from Abcam Inc. (Cambridgeshire, UK). Synthetic oligonucleotide siMiD51-690, siMiD51-858, siMiD51-1021 and miR-107 mimics, and negative controls were purchased from Genepharma (Suzhou, China). Goat anti-Rabbit IgG (H + L) and anti-Mouse IgG (H + L) secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA). A total cholesterol assay kit, triglyceride assay kit, high-density lipoprotein cholesterol assay kit and low-density lipoprotein cholesterol assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China).

Plasma of total cholesterol, HDL-C, ApoA-I, and LDL-C was detected total cholesterol assay kit, high-density lipoprotein cholesterol assay kit, Apolipoprotein A-I Assay Kit, and low-density lipoprotein cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute). The free cholesterol and total cholesterol in liver and cells were detected by gas chromatography–mass spectrometry (GC-MS). Liver tissues (10 mg–20 mg) and hepatocytes (1 × 10⁶) were homogenized in 1× PBS. Cholesterol was extracted at m/z = 329, 368, 458, and Internal standard (IS) was extracted at m/z = 217, 357 by GC chromatograms.

Traditional dark tea (first grade) was purchased from the tea market in Liyang, China. Probiotic dark tea was purchased from Longxing Ecological Agriculture Garden, Liyang, China. Normal diet pellets were purchased from Nanjing Anlimo Technology Co., Ltd. (Nanjing, China), while high-fat diet pellets (D12492, Rodent Diet with 60 kcal % Fat) were purchased from Shanghai Research Science Co., Ltd. (Shanghai, China). The total triglyceride assay kit, total cholesterol assay kit and high-density lipoprotein cholesterol assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Male Sprague-Dawley (SD) rats (four weeks old) weighing 80–100 g were purchased from the Experimental Animal Center of Jiangsu University (License No. SCXK (Su) 2013-0011, Yangzhou, China) and maintained at 22 ± 2 °C, 60 ± 10% humidity, natural light environment. The rats were fed with sterilized pellet diet and water ad libitum. The animals were acclimatized for two weeks under laboratory conditions. Ethical clearance for handling the animals was obtained from the ethical committee (Animal Care and Use Committee of Yangzhou University Approval ID: YDDW201606-002, Yangzhou, China) constituted for the purpose.

The levels of serum total cholesterol (TC), triglyceride (TG), glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were evaluated using the TG Triglyceride Kit (single agent GPO-PAP method, Jiancheng, Nanjing, China, A110-1-1), Total cholesterol assay kit (single agent GPO-PAP method, Jiancheng, Nanjing, China, A111-1-1), Alanine aminotransferase assay kit (Reitman Frankel assay, Jiancheng, Nanjing, China, C009-2-1), Aspartate aminotransferase assay kit (Jiancheng, Nanjing, China, C010-2-1), low-density lipoprotein cholesterol assay kit (Jiancheng, Nanjing, China, A113-1-1), and high-density lipoprotein cholesterol assay kit (Jiancheng, Nanjing, China, A112-1-1) respectively.

The blood samples were collected via retro‐orbital collection method, followed by coagulation at room temperature. Subsequently, the samples were centrifuged at 3500 g for 15 min to separate the serum. The concentrations of total cholesterol (T‐CHO), low‐density lipoprotein cholesterol (LDL‐C), and high‐density lipoprotein cholesterol (HDL‐C) were determined using the total cholesterol assay kit (Jiancheng, A111‐1), low‐density lipoprotein cholesterol assay kit (Jiancheng, A113‐1), and high‐density lipoprotein cholesterol assay kit (Jiancheng, A112‐1), respectively, following the manufacturer's instructions.

PDGF‐BB was purchased from PeproTech (100‐14B, Rocky Hill). GAPDH monoclonal antibodies (60004‐1‐Ig), KLF4 polyclonal antibodies (11880‐1‐AP) and STAT3 polyclonal antibodies (10253‐2‐AP) were purchased from Proteintech. Anti‐P‐STAT3 (Ser 727) Rabbit pAb (WL03346) were purchased from Wanleibio. MLCK monoclonal antibodies (ab76092), ML‐7 (ab120848), Y‐27632 dihydrochloride (ab120129) and 4,6‐diamidino‐2‐phenylindole (DAPI, ab104139) were obtained from Abcam Inc. The STAT3 inhibitor S3I‐201 was obtained from Selleck Chemicals (S1155). FITC‐conjugated α‐smooth muscle actin antibody (F3777) and Oil‐Red‐O solution (O1391) was purchased from Sigma‐Aldrich (St Louis, MO). Rhodamine conjugated goat anti‐rabbit IgG (H+L) (31670) was purchased from Thermo Scientific. Total cholesterol assay kit, Triglyceride assay kit, High‐density lipoprotein cholesterol assay kit, Low‐density lipoprotein cholesterol assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (China).