Glucagon elisa 10 μl kit

Adult mice at 2–4 months of age were fasted overnight for 16 h, then anesthetized using continuous isoflurane inhalation. Plasma glucose was measured from the tail tip using a Breeze 2 glucometer (Bayer, Mishawaka, IN, USA). Whole blood was then collected by cardiac puncture and transferred to heparinized tubes (Sarstedt, Nümbrecht, Germany), which were temporarily stored on ice, then centrifuged at 2,000×g for 5 min at 4 °C, and the plasma supernatant was stored at −20 °C. Plasma glucagon levels were measured using the Glucagon ELISA – 10 μL kit (Mercodia, Uppsala, Sweden), following the manufacturer's instructions and using technical duplicates.

Whole pancreas was collected from adult mice after overnight fast as described above, then immediately immersed in 15 mL TRIzol Reagent (ThermoFisher, Waltham, MA, USA), homogenized, snap-frozen in liquid nitrogen, and stored at −80 °C. RNA was later extracted from the aqueous phase per manufacturer's instructions using 0.2X chloroform, followed by isopropanol precipitation. RNA quality and concentration were analyzed with the RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). cDNA was synthesized from 1 μg of total RNA using SuperScript II Reverse Transcriptase (ThermoFisher, Waltham, MA, USA). qPCR was performed using the intron-spanning primers listed in Supplemental Table 3 and 2X Brilliant III SYBR Green QPCR Master Mix (Agilent, Santa Clara, CA, USA), with technical triplicates.
DNA and protein were extracted from the organic phase of the Trizol homogenate, per manufacturer's instructions, with modified protein resuspension based on [22] (link). Total protein content was measured using the Bio-Rad Protein Assay standard procedure for microtiter plates (Bio-Rad, Hercules, CA, USA) using technical triplicates. Pancreatic glucagon content was measured using the Glucagon ELISA – 10 μL kit (Mercodia, Uppsala, Sweden), following the manufacturer's instructions and using technical duplicates.

Mice (8 weeks) were individually housed and fasted for 6 h. Mice were injected with 0.75 U/kg of insulin (Novolin-R, Novo Nordisk, Princeton, NJ; i.p.). Tail blood was collected in microfuge tubes containing 2 μg/mL aprotinin at the indicated times, spun down at 3000 rpm for 15 min, and plasma glucagon levels were measured with a Glucagon 10 μL ELISA kit (Mercodia).