Ultra sensitive mouse elisa kit

10-week old female mice were fasted (5 h, Pur-O-Cel bedding) and then bled from the tail vein to collect fasted plasma. Two days later, mice were again fasted (14–16 h, Pur-O-Cel bedding) and then orally gavaged with 0.5g glucose/kg body weight of Ensure (Abbott, Columbus, Ohio) to measure insulin response to a mixed meal. 30 minutes after the gavage, blood was collected through a submandibular bleed. Serum insulin from fasted and fed blood was measured using a mouse ELISA (Crystal Chem Ultra Sensitive Mouse ELISA Kit).

Insulin was measured in plasma using an Ultra Sensitive Mouse Elisa kit as described by the manufacturer (Crystal Chem, Zaandam, The Netherlands).

Insulin was measured in duplicates using ultra-sensitive mouse ELISA kit (90080, Crystal Chem, Downers Grove, IL) according to manufacturer’s instructions.
Adiponectin was measured using a commercial available ELISA kit according to manufacturer’s protocol (ELM-Adiponectin, RayBiotech, Norcross, GA).
For liver triglycerides measurements, 50 mg liver was weighted and added 125 ul ethanolic KOH in microfuge tubes. Samples were incubated overnight and added 175 ul H₂O:EtOH (1:1), centrifuged 5 min at 5000 rpm and the supernantant was moved to new tubes. 100 ul EtOH was added, vortexed and 200 ul was moved to new tubes and added 215 ul 1M MgCl₂. Samples were centrifuged, moved to new tubes and measured for triglycerides.
Free fatty acids were measured by a commercial available kit according to the supplied instructions (NEFA-HA(2), Wako, Neuss, Germany).

In the final week, semi‐fasting blood glucose levels were measured by glucometer on tail blood after 5 h of fasting. After euthanizing, heparinized blood was collected, centrifuged, and plasma obtained for analysis. ELISAs were performed for insulin (Ultra‐Sensitive Mouse ELISA kit, Crystal Chem, Downers Grove, IL) and IGF1 (ab100695, Abcam, Cambridge, UK) according to the manufacturers’ instructions. Triglycerides were determined by a colorimetric assay (BioVision Incorporated, Milpitas, CA).

In vivo metabolic testing was performed as previously described [23 (link),36 (link),37 (link)], for glucose tolerance testing, mice were fasted for 12 hours, followed by intraperitoneal (IP) injection of 1 mg/kg glucose. For insulin tolerance testing [23 (link),36 (link),37 (link)], mice were fasted for 4 h, followed by IP injection of 0.75 units/kg insulin (Actrapid; NovoNordisk, Bagsvaerd, Denmark). Whole-blood glucose was determined at 30 min intervals by tail vein sampling using a portable metre (Accu-chek Aviva; Roche Diagnostics, Burgess Hill, U.K.). Plasma insulin was measured using ultrasensitive mouse ELISA kit (CrystalChem, Downers Grove, IL), as previously described [37 (link)]. To examine in vivo insulin signalling, after 3 weeks of cixutumumab treatment, fasted mice were injected IP with insulin (0.75 U/kg) or vehicle (saline). After 15 min, mice were sacrificed and livers rapidly harvested and snap-frozen.