Gentamicin sulfate

USP1 mouse embryonic stem cells (mESCs) transfected with enhanced green fluorescent protein (eGFP) with retroviral vector (Paulsen et al., 2011 (link)) were maintained on a cell-free murine embryonic fibroblast-based substrate to avoid fibroblast contamination between cell passages (Stelling et al., 2013 (link)). Cells were maintained in D-MEM/F12 (Gibco) supplemented with 15% fetal bovine serum (JRH Biosciences), 2 mM glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 40 µg/mL gentamicin sulfate (Schering-Plough), and 0.2% of conditioned medium of CHO (Chinese hamster ovary) cells producing leukemia inhibitory factor (LIF).

The organs were isolated macerated and the cells adjusted in concentration 1 × 10⁶ cells/well in RPMI culture (Hyclone, Logan, UT, United States) supplemented with 10% fetal bovine serum (CULTILAB, Campinas, SP, Brazil), 2 mM of l-glutamine (Gibco-BRL, Life Technologies, Grand Island, NY, MO, United States), 25 mM HEPES (Sigma, St. Louis, MO, United States), 50 μM 2-mercaptoethanol (Pharmacia Biotech, Uppsala, Switzerland) and 20 μg/mL gentamicin sulfate (Schering-Plough, Rio de Janeiro). To evaluate production of cytokines, cells were stimulated with 50 μg/mL CII or 10 μg/mL mBSA. A positive control to each sample was stimulated with 1 μg/mL anti-CD3 antibody (Bioscience) and a negative without stimulation. After 48 h in a CO₂ incubator the supernatants were collected for measurement of IL-17, IFN-γ by ELISA.

Infection was assessed by counting the number of Colony Forming Units (CFUs) of P. brasiliensis recovered from infected mice’s lungs. Four animals from each group were euthanized by CO₂ chamber at indicated time points, and the lungs were aseptically collected. One longitudinal section of each lung was weighed and macerated within sterilized PBS. One hundred µl from the homogenized lung tissues was plated into BHI agar supplemented with 4% horse serum; 5% P. brasiliensis 192 isolate yeast culture filtrated supernatant, and 40 mg/L of gentamicin (Gentamicin Sulfate, Schering-Plough, Rio de Janeiro, Brazil). Plates were incubated for seven days at 37°C, and CFUs were counted.