Registered nurses drew 10 ml of peripheral venous blood from each participant at both the pre- and post-assessment and stored the samples in BD Vacutainer SST tubes (367955, BD Diagnostics, Franklin Lakes NJ, USA). After clotting, we separated the serum samples from whole blood samples by centrifugation at 3,000 g for 5 min at 18 °C and then stored them in four 1.5-ml aliquots at −80 °C. We measured serum IL-6 by commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (HS600C, R&D Systems, USA). We assayed all samples in duplicate and then log-transformed participants’ IL-6 levels to obtain the normal distribution for analysis.
Hs600c
Manufactured by R&D Systems
Sourced in United States
The HS600C is a benchtop centrifuge designed for a variety of laboratory applications. It features a fixed-angle rotor capable of holding up to 6 tubes with a maximum speed of 6,000 RPM. The HS600C is a compact and sturdy centrifuge suitable for general-purpose use in research and diagnostic settings.
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7 protocols using hs600c
1
Measuring Serum IL-6 Levels
2
Fasted Biomarker Analysis Protocol
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Fasted blood samples were collected from a cubital vein into vacutainers containing lithium heparin, EDTA, or Silica Clot Activator and centrifuged (1500 rpm for 15 min at 4°C), and the supernatant was stored at −20°C until later analysis. Plasma was analyzed for high-sensitive C-reactive protein (hsCRP; primary outcome), triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol (HITACHI/Roche COBAS 800 c502, Roche Diagnostics, Indianapolis, IN). Serum was analyzed for insulin (COBAS 8000, HITACHI/Roche, Tokyo, Japan) and interleukin (IL)-6 (primary outcome), in triplicate, with a commercially available ELISA (HS-600C, R&D, Minneapolis, MN). Fasted capillary blood samples from the earlobe were immediately analyzed for blood glucose levels (Glucomen Lx plus-meter with GlucoMen LX sensor strips, Menarini Diagnostics, Firenze, Italy). HOMA-IR was calculated as fasting insulin (mIU/L) × fasting glucose (mg/dL)/405.
3
Cytokine and Adhesion Factor Secretion
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The secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), soluble intracellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in 24-h culture supernatants was evaluated by ELISA assays (R&D quantikine HS600C and HS800 for human IL-6HS and IL-8HS, DVC00 and DCD540 for human VCAM-1 and ICAM-1) or using the ELLA system (Protein Simple, Simple Plex Cartridge for IL-6 and Simple Plex Cartridge for IL-8, Bio Techne, Minneapolis, MN, USA). Cells were incubated for 14 days with/without the drugs. The culture medium was replaced for the last 24 hours by a medium without foetal calf serum with/without the drugs and collected after 24 hours to measure the level of secreted cytokines and adhesion factors.
4
Cytokine Levels in CM and Serum
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5
Measuring IL6 Secretion in HEK293T Cells
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Media from HEK293T cells transfected with for 48 hours with CRISPRa (VPR) alongside either SG-286 or trcrRNA constructs, were recovered. Following a 2 min centrifugation at 2000 x g, the cleared supernatants were frozen at -20 C until further analysis. Media were then thawed and assayed for IL6 using a commercial ELISA kit (HS600C, RandD systems) according to the supplier’s protocol.
6
Biochemical Analyses of Metabolic Markers
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Plasma glucose concentrations during the OGTT were measured at the department of Clinical Biochemistry at Rigshospitalet, Copenhagen, Denmark, using an ultraviolet test (Roche/Hitachi MODULAR P, Cobas, Roche, Basel, Switzerland). MSD kits were used to analyze insulin, leptin, and TNF-α concentrations in plasma (Adipokine Comb 1 hu, catalog no. K15276) and plasma glucagon concentrations were measured using radioimmunoassay kits (Millipore Cat# GL-32K, RRID:AB_2757819 ). Plasma concentrations of intact GIP and total GLP-1 were analyzed after extraction in 70% ethanol (vol/vol; final concentration). Intact, biologically active GIP was measured using antiserum code no. 98171 [33 (link)]. Plasma concentration of GLP-1 was measured against standards of synthetic GLP-1 7-36 amide using antiserum code no. 89390 in a final dilution of 1:200 000 and a tracer of I125-labeled GLP-1 (7-36 amide) as described in detail previously [34 (link)]. This antiserum is specific for the amidated COOH-terminus of GLP-1 and therefore mainly reacts with amidated GLP-1 of intestinal origin [34 (link)]. The assay reacts equally with intact GLP-1 and with the primary metabolite GLP-1 (9-36 amide) [34 (link)]. Plasma levels of IL-6 were measured using an ELISA (R&D Systems Cat# HS600C, RRID:AB_2893335 ).
7
High Sensitivity IL-6 and TNF-α ELISA
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