Crl cd1 icr mice

Male Crl:WI (Han) rats and Crl:CD1 (ICR) mice (6–8 weeks) from Charles River Laboratories (Lyon, France) were acclimatised for 1 week. Six animals were housed per cage, on a 12 h light/dark cycle, at constant temperature (22 ± 2 °C). Standard food and tap water were provided ad libitum. Care of animals was undertaken in compliance with the European Community Directive 86/609/CEE for the use of laboratory animals and with the Autonomous Catalan law (Decret 214/1997). All experimental procedures were approved by the Almirall Ethics Committee.

C57Bl/6 (B6) and B6.SJL-H2s (B6.s) mice were obtained from colonies held at the animal facility at the University of Lübeck. Mice were housed under specific pathogen-free conditions and provided standard mouse chow and acidified drinking water ad libitum. Mice aged 6–10 weeks were used for the experiments. All clinical examinations, biopsies, and bleedings were performed under anesthesia with i.p. administration of a mixture of ketamine (100 µg/g) and xylazine (15 µg/g). Evaluation of skin lesions was performed as described (10 (link)). Animal experiments were approved by local authorities of the Animal Care and Use Committee (Kiel, Germany) and performed by certified personnel. For KLH immunization studies, male Crl:CD1 (ICR) mice were purchased from Charles River. Female MRL/MpJ-Faslpr/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in polycarbonate cages, with free access to water and non-purified stock diet and allowed to condition for 2 weeks in their new environment at 22 ± 2°C, 40–70% relative humidity, and 12 h:12 h light:dark cycles. All animal care and experimental procedures followed the European Community Directive 86/609/CEE and the Autonomous Catalan law (Decret 214/1997) for the use of laboratory animals and were approved by the Almirall Animal Experimentation Ethical Committee.

Islets were isolated from 10–12-week-old male Crl:CD1 (ICR) mice (Charles River), by collagenase digestion of the exocrine pancreas [15 (link)]. Human islets were isolated from heart-beating non-diabetic donors, with appropriate ethical approval, at the King’s College London Human Islet Isolation Unit [16 (link)]. Islets were maintained overnight at 37 °C in culture medium supplemented with 5.6 mM glucose, 10% FCS, 2 mM glutamine and penicillin–streptomycin (100 U/mL, 0.1 mg/mL) before experimental use.

N2aC24 and N2aC24L1-3 cells [15 (link)] were cultured at 37 °C with 5% CO₂ in air in classic DMEM (Wako Pure Chemical Industries) with 10% FBS (Gibco Life Technologies Corporation) or in advanced DMEM (Gibco Life Technologies Corporation) with 2% FBS (Gibco Life Technologies Corporation) supplemented with 1× Penicillin-Streptomycin Mixed Solution (26253-84, Nacalai Tesque). Crl:CD1(ICR) mice were purchased from Charles River Laboratories Japan (Kanagawa, Japan).

Toxicity of BoNT in mouse local muscle was assessed as previously reported [37 (link),38 (link)]. Female Crl: CD1(ICR) mice were purchased from Charles River Japan. Mice weighing 20–30 g were anesthetized with isoflurane and injected with BoNTs in 10 µL of 0.2% gelatin-phosphate buffer (pH 6.3) in the right hind limb muscle. Mice were suspended by the tail briefly, and the degree of digit abduction of the leg was scored on a scale of 0 (normal) to 4 (maximum paralysis). The paralysis scores, body weights, and clinical scores (Table S1) of the mice were monitored at least two times per day for 4 d, once per day for 7 d, and then once every other day. The humane endpoint was set as a total clinical score of above 5.