Glucose colorimetric assay kit

Blood was collected from 32-week-old chow-fed mice (thermoneutrality) or 12- to 16-week-old high-fat diet-fed mice (standard housing temperatures) fasted for 5 h by cardiac puncture. Blood was collected and inverted in SST™ amber tubes (BD Microtainer^®) and incubated at room temperature for 30 min. Samples were then centrifuged at 12,000 g for 10 min and the separated serum collected. Insulin, adiponectin, leptin, AST and ALT analysis was performed at the Core Biochemical Assay Laboratory (Cambridge, UK). Glucose levels were determined using a Glucose Colorimetric Assay Kit (Cayman Chemical), following the manufacturer's protocol provided. Serum triglyceride levels were determined using a Triglyceride Liquid Assay (Sentinel Diagnostics), following the manufacturer's instructions. QUICKI was calculated from fasting glucose (mg/dl) and insulin (µU/ml) values as previously described (Katz et al., 2000 (link)). QUICKI=1/[log(I₀)+log(G₀)], where I₀ is fasting insulin and G₀ is fasting glucose. QUICKI is a dimensionless index without units.

Medium samples were diluted 10-fold in PBS. Glucose concentration was determined using the Glucose Colorimetric Assay Kit (Cayman) according to the manufacturer’s protocol. Standards were prepared in PBS.

Glucose levels were quantified using a glucose colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. The standard curve was prepared with the provided glucose that was serially-diluted. Absorbance was measured at 520 nm using a Synergy HT microplate spectrophotometer (BioTek, Winooski, VT), where all samples were assayed in triplicate.

To measure the glucose concentration in yeast media, a single colony of CM018, CM019 and CM022 was inoculated in 5 ml YP 2% glucose media and allowed to grow until OD_{600 nm} reached 0.5. Cells were then inoculated into 50 ml YPD cultures with 2%, 0.5% or 0.2% glucose at an initial OD_{600 nm} of 0.0005. 0.2 ml media samples removed at indicated cell densities were centrifuged at 800 g for 10 min at 4°C to pellet yeast cells. Supernatants were then transferred to new tubes and stored at −20°C until measurement. Glucose concentration was assayed by using a Glucose Colorimetric Assay Kit (Cayman Chemical) per the manufacturer's protocol.

Plasma samples from fasted mice stored at −80 °C were assayed for glucose concentration with the Glucose Colorimetric Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Plasma samples were diluted 1:10 with an assay diluent prior to using the standard protocol, utilising a standard curve run in parallel. The absorbance of 514 nm was read on an EnSpire Multimode Plate Reader (PerkinElmer Australia, Melbourne, VIC, Australia).