Nystatin suspension

One gramme of fresh faecal sample per infant was serially diluted in maximum recovery diluent (Fluka, Sigma Aldrich, Ireland). Enumeration of bifidobacteria was performed by spread-plating serial dilutions onto de Man, Rogosa, Sharpe agar (Difco, Becton-Dickinson Ltd., Ireland) supplemented with 0.05% L-cysteine hydrochloride (Sigma Aldrich), 100 μg/ml mupirocin (Oxoid, Fannin, Ireland ) and 50 units nystatin suspension (Sigma Aldrich). Agar plates were incubated anaerobically at 37 °C for 72 h (Anaerocult A gas packs, Merck, Ocon Chemicals, Ireland). Enumeration of lactobacilli was determined by plating samples onto Lactobacillus selective agar (Difco) with 50 units nystatin and incubated anaerobically at 37 °C for 5 days. Bacterial counts were recorded as colony forming units (CFU) per gram of faeces and were log10 transformed prior to statistical analyses.

Tissue preparation, immunostaining, and semiquantitative evaluation were done as described.⁴⁰ (link) Lymphoblastoid cell lines were maintained after EBV immortalization. Fibroblasts were cultured from a 3 mm punch skin biopsy. In addition to these cells, we also purchased a control immortalized PBMC (ATCC #CRL5959) of a normal individual (a 58-year-old male, European descent) and two control primary fibroblasts (Coriell #GM07753 and #GM07492) of apparently healthy individuals (both are 17-year-old males, European descent, named CTL-17Ma and -17Mb, respectively). All the immortalized PBMCs were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 15% fetal bovine serum (FBS) and 4 mM L-Glutamine (Thermo Scientific). For prevention against bacteria, fungi, and mycoplasma, the media also contained Penn Strep (Thermo Scientific), Normocin (Invivogen), Nystatin suspension, and Amphotericin B solution (Sigma Aldrich) according to manufacturers’ instructions. The primary fibroblasts were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) containing high glucose and GlutaMAX (Thermo Scientific) and supplemented with 15% FBS and 1% Penn Strep. Cells were incubated at 37°C in a humidified CO₂ incubator. The immortalized PBMCs of less than 30 passages and primary fibroblasts of less than 20 passages were employed in the experiments.