Skin punch biopsies (4 mm) were collected from the dorsal skin of mice immediately after euthanasia. Biopsies were incubated in DMEM plus antibiotics with 2 mM L-glutamine and 10 mM HEPES (Thermo Fisher) for 24 h at 37°C in a 5% CO2 incubator. The conditioned media was snap-frozen and stored at −80°C until analyzed.
Whole blood was collected by cardiac puncture into EDTA tubes (T-MQK, Terumo Medical Corp., Somerset, NJ, USA). Plasma was separated by centrifugation of whole blood at 1,200 g and 4°C for 10 min. Equal volumes of plasma and methanol were added to a 1.5 ml microcentrifuge tube, mixed, and incubated on ice for 3 min. Supernatant was collected after the mixture was centrifuged at 5,000 g for 5 min. The extract was then dried in a centrifugal concentrator for 2–3 h at room temperature and reconstituted with assay buffer prior to analysis.
The processed sample was analyzed using the Histamine ELISA kit (ENZ-KIT140-0001, Enzo LifeScience). Assay results were collected and calculated using the Synergy HT plate reader and Gen5™ 1.0 data analysis software (BioTek).
Whole blood was collected by cardiac puncture into EDTA tubes (T-MQK, Terumo Medical Corp., Somerset, NJ, USA). Plasma was separated by centrifugation of whole blood at 1,200 g and 4°C for 10 min. Equal volumes of plasma and methanol were added to a 1.5 ml microcentrifuge tube, mixed, and incubated on ice for 3 min. Supernatant was collected after the mixture was centrifuged at 5,000 g for 5 min. The extract was then dried in a centrifugal concentrator for 2–3 h at room temperature and reconstituted with assay buffer prior to analysis.
The processed sample was analyzed using the Histamine ELISA kit (ENZ-KIT140-0001, Enzo LifeScience). Assay results were collected and calculated using the Synergy HT plate reader and Gen5™ 1.0 data analysis software (BioTek).