Alphalisa immunoassay detection kit

Glucose tolerance tests (GTT) were performed on overnight-fasted mice, and insulin tolerance tests were performed on 4 h fasted mice. Mice received an oral gavage bolus of D-glucose (2 mg/g) or intraperitoneal injection with insulin (0.6 IU/kg), and subsequently tail blood glucose was measured (Accu-chek performa; Roche, Basel, Switzerland) at the time points indicated [24 (link)]. The meal challenge experiments involved fasting mice overnight (16 h) then allowing ad libitum access to food for 3 h, before re-fasting for 4 h and monitoring blood glucose at baseline, after 1 and 3 h of refeeding, and then 2 and 4 h of fasting. Blood samples were collected from a tail vein in the fasted state and from fed mice after access to diet for 1 h. Plasma ALT, triglycerides and non-esterified fatty acids (NEFA) were measured by an autoanalyzer (Cobas Mira; Hoffmann-La Roche, Basel, Switzerland) and insulin with AlphaLISA immunoassay detection kit (PerkinElmer, Waltham, MA, USA). Plasma insulin and blood glucose levels were used to calculate homeostatic model assessment of insulin resistance (HOMA-IR) values using the formula from Matthews et al. [25 (link)].

Following the 6-week intervention period, blood samples were collected from a tail vein following an overnight fast (fasted) and when re-fed ad libitum for 1 h (fed). Blood glucose was determined using a hand-held glucose meter (Accu-Chek Performa; Roche, Basel, Switzerland) and plasma insulin with AlphaLISA immunoassay detection kit (PerkinElmer, Waltham, MA). Mice were killed by cervical dislocation and a small portion of liver and skeletal muscle (gastrocnemius) was placed in organ transplant buffer (custodiol HTK) to measure mitochondrial function, with the remaining tissue being snap-frozen in liquid nitrogen and stored at −80°C until analysis.