Medetomidine hydrochloride

All experiments were conducted in accordance with the ethical standards of our university (Animal Care Committee of Kanazawa University, AP-183983) and with international standards for animal welfare and institutional guidelines. EC-14 was cultured in Luria–Bertani broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for 12 h and then seeded into new broth and incubated at 37 °C for 12–14 h with shaking. Subsequently, EC-14 was collected by centrifugation at 8000× g for 5 min at 4 °C and suspended for inoculation into mice. Male Jcl:ICR mice (n = 4) (4 weeks old; CLEA Japan, Tokyo, Japan) were purchased 7 days prior to the experiments and subjected to immunosuppression treatment with 150 mg/kg and 100 mg/kg of endoxan (Shionogi) at 4 days and 1 day prior to infection, respectively.
EC-14 (approximately 5 × 10⁶ CFU/100 µL) was injected into the muscle of the hind leg of mice under anesthesia. Mice were anesthetized using a mixture of butorphanol tartrate, midazolam, and medetomidine hydrochloride (Fujifilm Wako Pure Chemical Corp., Osaka, Japan). Mice were euthanized at 2, 6, 8 and 24 h after infection. Subsequently, the infected muscle was collected and homogenized in PBS. The number of CFU in the homogenized samples was determined by dilution in PBS and plating on agar medium.

PRX-VBn consisting of α-CDs and PEG (M_n = 5000) capped with 4-vinylbenzyl groups was synthesized as previously described by Arisaka et al. [13 (link)] (Figures S2 and S3 in Supporting Information). Iodomethane (MeI) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Methanol (MeOH) was purchased from Kanto Chemical (Tokyo, Japan). Collagen sponges for 35 mm culture dishes were purchased from Koken (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Gibco BRL, Life Technologies (Carlsbad, CA, USA). Ammonium persulfate (APS), butorphanol tartrate, dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (high glucose, with L-glutamine, phenol red, and sodium pyruvate) (D-MEM), 100 U/mL penicillin, 100 µg/mL streptomycin, medetomidine hydrochloride, midazolam, phosphate buffered saline (PBS), sodium hydroxide (NaOH) and N,N,N’,N’-tetramethylene diamine (TEMED) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The BALB/3T3 clone A31, a fibroblast cell line established from mouse embryos, was obtained from the American Type Culture Collection (Manassas, VA, USA). BALB/c mice were obtained from Sankyo Labo Service (Tokyo, Japan).