In vivo experiments in humanized Alb−/−FcRn−/−hFcRn+/+ mice

In Vivo Experiments in Humanized Alb^-/-FcRn^-/-hFcRn^+/+ mice

Humanized male Alb^-/-FcRn^-/-hFcRn^+/+ (Cg-Tg(FCGRT)32Dcr Alb^em12Mvw Fcgrt^tm1Dcr/MvwJ mice, The Jackson Laboratory) and wild-type C57BL/6J mice were housed on woodchip bedding in cages with 50–60% humidity and a 12 hour light/dark cycle and given ad libitum access to food and water. To test the effects of HSA infusions, two HSA doses of 1.5 g/kg b.w. or HBSS^-- were administered 72 and 24 hours prior to administer an isoflurane overdose to collect peritoneal exudates by peritoneal lavage with 7 ml of ice-cold DPBS--. To isolate peritoneal macrophages, exudates were centrifuged at 500 g for 5 min and the pellet resuspended in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM) and 5% FBS. Cells were seeded in 24-well plates and allowed to adhere in a humidified 5% CO₂ incubator at 37°C during 2 hours. Non-adherent cells were removed by washing twice with DPBS--. In some experiments, peritoneal macrophages from wild-type and humanized Alb^-/-FcRn^-/-hFcRn^+/+ mice were treated with either vehicle, CpG-DNA (4 µM) or CpG-DNA plus HSA (15 mg/ml) for 2 hours. All studies were conducted in accordance with the criteria of the Investigation and Ethics Committee of the Universitat de Barcelona (UB) (protocol number: 139/19) and the European Community laws governing the use of experimental animals.