Neomycin sulfate

The human colonic epithelial cell line Caco2-C2BBe1 was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 50 μg/mL penicillin, 50 μg/mL streptomycin sulfate, and 100 μg/mL neomycin sulfate (Invitrogen, Carlsbad, CA, USA). An intestinal epithelial monolayer was formed as per the method of Chen et al. [19 (link)]. Briefly, the cells were cultured under a humidified atmosphere of 5% CO₂ at 37 °C. The Caco-2 cells were seeded onto permeable 12-well Transwell membranes (Corning, Lowell, MA) with a 3-μm pore size (density, 10⁵/cm²) to form the intestinal epithelial monolayer. The culture medium was replaced with fresh medium every 2 days during the 28-day culture period. After 28 days, the transepithelial electrical resistance (TEER) of Caco-2 epithelial monolayers was >300 Ω·cm², indicating that the monolayers were ready to use [20 (link)]. The TEER was measured using an epithelial volt-ohmmeter with an STX2 probe (World Precision Instruments, Sarasota, FL, USA).

Mice were provided autoclaved drinking water supplemented with ampicillin (1 g/L, Sigma-Aldrich, Madrid, Spain), metronidazole (1 g/L, Sigma-Aldrich, Madrid, Spain), neomycin sulfate (1 g/L, Gibco-Invitrogen, Barcelona, Spain) and vancomycin (0.5 g/L, Sigma-Aldrich Madrid, Spain). ABX treatment started 14 days before TMEV infection and terminated on day 14 post-infection (pi). Animals were sacrificed on day 14 or 85 pi to assess the influence of early ABX treatment on the outcome and severity of the disease.

A murine transgenic αPIG-iPS cell line, in which the puromycin-N-acetyl transferase and the enhanced green fluorescent protein (eGFP) genes are under the control of the cardiac-specific alpha-myosin heavy chain (α-MHC) promoter [41 (link)], was used in this study to facilitate bioprocess development. The fluorescence marker and the antibiotic resistance genes, specifically expressed in CMs, allow easy monitoring of the cardiac differentiation process and selection of a highly pure CM population upon addition of puromycin into the media, respectively. iPSCs were cultivated on a monolayer of mitotically inactivated murine embryonic fibroblasts (MEFs) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 15 % (v/v) fetal bovine serum (FBS), 1 % (v/v) non-essential amino acids (NEAA), 2 mM L-glutamine, 50 μM β-mercaptoethanol, 500 μg/mL neomycin sulfate (all from Invitrogen, UK), and 1000 U/mL leukemia inhibitory factor (LIF) (ESGRO, Merck Millipore, Germany), at 37 ºC in a humidified atmosphere of 5 % CO₂. Cells were passaged every two days as previously reported [41 (link)].

Act-mOVA mice (CD90.2) were lethally irradiated with 12 Gy and reconstituted with T cell-depleted bone marrow from either C-myc^fl/fl OT1 or C-myc^fl/flCd4^Cre-Tg OT1 (CD90.1) mice. All irradiated mice received antibiotics in the drinking water (1 g/l neomycin sulfate, Thermo Fisher Scientific) during the first 3 weeks after irradiation.