Plasma ACTH Assay in Mice

Plasma ACTH assays were performed as described previously^{11 (link)}. Briefly, after mice were killed by cervical dislocation and decapitation, trunk blood was collected directly into blood collection tubes (Becton Dickinson) containing 50 μL aprotinin (Phoenix Pharmaceuticals). Plasma was obtained by centrifugation at 1600×g for 15 min at 4 °C, and stored at − 80 °C. Plasma ACTH concentrations were measured using the ACTH ELISA kit (MD Biosciences), according to the manufacturer’s instructions, with the following modifications: (1) 100 μL of the controls or blood plasma combined with 100 μL of PBS (Phosphate Buffered Saline, pH 7.4) was used in the place of 200 μL plasma and (2) the results were assessed with the QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher). Fluorescence was measured with a CytoFluor4000 plate reader (Applied Biosystems). ACTH assays were carried out in duplicate. The average inter- and intra-assay coefficients of variation were 4.0% and 5.8%, respectively.