Emd millipore milliplex map immunoassay

Blood samples were taken by a qualified nurse before and 72 h after the last intervention. Strenuous exercise, caffeine, and alcohol were restricted 48 h before blood drawing. After 12 h of fasting, participants were required to arrive at the lab before 7:30 am to take a 5-mL blood sample from the cubital vein. After 1-h coagulation at room temperature (22 °C), serum was separated by centrifugation (3000 rpm for 5 min) and immediately stored at −80 °C for later analysis.
To minimize the inter-assay variation, blood tests were conducted at the end of the study by KingMed Diagnostics (Limited company, Guangzhou, China) in standard procedures according to the manufacturer’s instructions. Fasting glucose was assessed using an automatic biochemical analyzer (Olympus AU400, Olympus, Japan). Metabolic regulating hormones, including insulin, C-peptide, glucagon, leptin, ghrelin, and gastric inhibitory peptide (GIP), were measured using the EMD Millipore Milliplex MAP immunoassay (Merck KGaA, Darmstadt, Germany). Insulin sensitivity was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR) index, calculating as: fasting serum insulin (μIU·mL⁻¹) × fasting serum glucose (mmol·L⁻¹)/22.5. The coefficients of variations for intra-assay and inter-assay were less than 3%.

Blood samples were collected at the same phase of each subject’s menstrual cycle (i.e., the luteal phase) at different measurement time points. Strenuous physical activity, caffeine, and alcohol were prohibited for 48 h before blood sample collection. Subjects arrived at the laboratory at around 7 a.m. under the condition of fasting overnight (>10 h), and 5 ml blood was drawn from the cubital vein by a certificated nurse using a serum separation tube. The blood samples were left for clotting at room temperature for 1 h and then centrifuged at 3000 rpm for 5 min; serum was separated and immediately frozen at −80°C for later analysis.
Leptin, ghrelin, tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were measured using the EMD Millipore Milliplex MAP immunoassay (Merck KGaA, Darmstadt, Germany). All blood samples were measured in standard procedures in accordance with the manufacturer’s instructions (KingMed Diagnostics Co., Ltd., Guangzhou, China), and were conducted at the end of the study to minimize variability.