Ilab 650 clinical analyser

Basal and stimulated lipolysis was performed on in vitro differentiated cells in 12-well plates as follows: 2 days before lipolysis experiment, differentiated cells were incubated in basal media containing either 0.05 µg ml⁻¹ doxycycline or vehicle. On treatment day, cells underwent a 2-h wash out period in Krebs Ringer HEPES (KRH) buffer containing 5 mM glucose and 3.5% bovine serum albumin (BSA), then incubated for 2 h in fresh KRH buffer (for basal lipolysis) or KRH buffer containing 10 nM isoproterenol (for stimulated lipolysis). At the end of the 2-h period, the treatment buffer was collected for glycerol measurements, and cells harvested in lysis buffer containing 1% IGEPAL CA-630, 150 mM NaCl and 50 mM Tris-HCl pH 8.0 for protein. Glycerol concentrations were measured using the GY105 enzymatic assay (Randox Laboratories Ltd) on an ILAB 650 clinical analyser (Instrumentation Laboratory UK) and normalised to cellular protein concentration.

As described previously (28 (link)), plasma samples were assayed for glucose and triacylglycerol (TAG) using ILab 650 Clinical Analyser (Instrumentation Laboratory, North Risley, UK). The ILab was calibrated before every experimental run using standard ReferrIL G (Instrumentation Laboratory).
The hormones (insulin, C-peptide, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (total), glucagon, ghrelin, leptin, resistin, visfatin and plasminogen activator inhibitor-1 (PAI-1)) were measured with a multiplex immunoassay (Bio-Plex Pro™ Human Diabetes Assay, Bio-Rad Laboratories Ltd) requiring 50 µL of plasma per sample. The BioPlex software (version 6.1) automatically generated the standard curve using Brendan Scientific's 5-Parametric Logistic Regression. The analyses took place over nine separate runs. All the samples from each participant were measured in the same assay and the intra-assay % CV ranged from 2.5% to 13.2% (n = 3). Missing data from a combination of sources (missing samples, undetectable, outliers) were 5.6%. Each assay plate had at least one person from each of the three study groups.

At the end of the experimental period (day 40), pancreatic tissues were excised for analysis of insulin content [36 (link),37 (link)]. Blood was taken from fasted mice for measurement of lipid profiles including HDL-cholesterol, LDL-cholesterol and triglyceride levels by an ILab 650 Clinical Analyser (Instrumentation Laboratory, Warrington, UK). Amylase activity (Amylase assay kit, Abcam, UK) and circulating total GLP-1 (ELISA, Millipore, UK) concentrations was measured as described in manufacturer’s protocol [38 (link)].