Amphotericin b

HiMCs were isolated from surgical tissue from patients who underwent bowel resection, as previously described [49 (link)]. Permission to conduct the study was obtained by the local ethical committee. Tissue underwent mechanical shredding and enzymatic digestion with pronase (Serva, Heidelberg, Germany) and collagenase D (Nordmark Biochemicals, Uetersen, Germany). After overnight culture of obtained cell suspension in RPMI 1640+GlutaMax^TM (Gibco Invitrogen, Paisley, OR, USA) with 10% FBS (Merck, Darmstadt, Germany), 100 μg/mL streptomycin, 100 U/mL penicillin (HyClone^TM Laboratories, South Logan, Utah, USA), 100 μg/mL gentamycin, and 2.5 μg/mL amphotericin B (CarlRoth Karlsruhe, Germany), enrichment of cells by magnetic cell separation of c-Kit+ (CD117) cells was conducted using CD117 microbead kit after dead cell removal kit (MACS^TM system, Miltenyi Biotech, Bergisch Gladbach, Germany). Pure hiMCs were cultured with 25 ng/mL stem cell factor (SCF) (PeproTech, Hamburg, Germany) and 2 ng/mL IL-4 (PeproTech) for at least 14 days before use in experiments.

The mixture of colon and small intestine homogenates (n = 3–5 mice per group) were plated on Middlebrook 7H10 Agar (Difco, Heidelberg, Germany) containing Mycobactin J (IDVet Innovative Technology, Montpellier, supplemented with antibiotics [Vancomycin (Roth, Karlsruhe, Germany); Amphotericin B (Roth); and Nalidixin Acid (Sigma, Munich, Germany)]. The plates were incubated at 37°C for up to 8 weeks.

Acetic acid, agar-agar, crystal violet, 3-(N-morpholino)propanesulfonic acid (MOPS), peptone, and yeast extract were obtained from Carl Roth GmbH (Karlsruhe, Germany). RPMI-1640 medium supplemented with L-glutamine was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluconazole was obtained from Merck KgaA (Darmstadt, Germany), amphotericin B—from Carl Roth GmbH (Karlsruhe, Germany). Each of Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) (10% (w/v)), and penicillin–streptomycin (100 U*mL⁻¹, 1% (w/v)), as well as Accutase^® and Eagle’s minimum essential medium non-essential amino acids (MEM NEAAs) were obtained from Life Technologies (Carlsbad, CA, USA). Phosphate-buffered saline (DPBS) was also sourced from Life Technologies (Carlsbad, CA, USA).