Blood was collected via cardiac puncture from 8- to 10-week-old k18-hACE2 mice that received SARS-CoV-2 of 4 × 105 TCID50/50mL/mouse via intranasal administration under general anesthesia (3.5% isoflurane). The animals were nebulized with rACE2 (deliver dose 12 mg/kg) from 2dpi until the day before sacrifice. The control group received PBS nebulization from 2dpi until the day of sacrifice. Mice were euthanized on 6 or 7 dpi under anesthesia by 3–5% isoflurane and in tubes containing heparin as an anticoagulant. Blood was immediately centrifuged at 1,500 × g for 10 min at 4°C, and the resulting supernatant (plasma) was collected. The RAGE concentration was measured using an ELISA kit (Abcam, ab190807).
Lung tissue homogenates were centrifuged for 15 min at 13,000 rpm 4°C, and the collected supernatants were used to measure the concentration of 17β estradiol (Abcam, ab108667), hACE2 (Abcam, ab235649), and Estrogen receptor α (Abbexa Ltd, abx254060) using ELISA kits according to manufacturer’s instructions.
Lung tissue homogenates were centrifuged for 15 min at 13,000 rpm 4°C, and the collected supernatants were used to measure the concentration of 17β estradiol (Abcam, ab108667), hACE2 (Abcam, ab235649), and Estrogen receptor α (Abbexa Ltd, abx254060) using ELISA kits according to manufacturer’s instructions.