75 cm2 tissue culture flask

The HT-29 human colorectal adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC #HTB-38) and was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.05 mg/mL gentamicin (Sigma-Aldrich, MO, USA) and were grown in 75 cm² tissue culture flasks (Greiner-Bio-One, NC, USA) at 37°C, 5% CO₂ in a humidified incubator (Thermo Fisher, MA, USA). Cells were passaged every 3 days or when cells reached ~90% confluency. Cell passages 12–28 were used for all assays.
The non-transformed rat IEC-6 (ATCC #CRL-1592) small IEC line was maintained in DMEM supplemented with 10% FBS and 0.05 mg/mL gentamicin, and cell cultures were maintained in a similar manner to HT-29 cells. Cell passages 6–15 were used for all assays.

The human gastric adenocarcinoma AGS cell line, was obtained from the American Type Culture Collection. Cells were maintained in Ham’s F12 medium (Lonza) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-Glutamine and 1% (v/v) non-essential amino acids. Cells were routinely cultured in 75 cm² tissue culture flasks (Greiner) at 37 °C and 5% CO₂ in a humidified atmosphere.

All human tissue was procured with written informed consent from the donor or next of kin and with ethical approval from the North of Scotland Research Ethics Committee (REC reference number 10/S0802/12). Human islets were isolated from brain-dead adult donor pancreata at the Scottish Islet Isolation Laboratory, Edinburgh, UK, under GMP conditions. Islet-enriched fractions were plated at a density of 3 x 10⁵ clusters on 75 cm² tissue culture flasks (Greiner, Stonehouse, UK) and cultured in serum-containing medium (SCM) prepared using RPMI 1640 (Gibco, Life Technologies, Paisley, UK) supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate (all from Gibco), and 75 μM β-mercaptoethanol (Sigma Aldrich, Dorset, UK). Cells were passaged every 5–7 days using trypsin (0.05%) and EDTA (0.02%, Gibco). Serum-free medium (SFM) was prepared with RPMI supplemented with 1% BSA and 10 μg/ml insulin, 5.5 μg/ml transferrin and 6.7 ng/ml sodium selenite.

Hs766t cells (ATCC, Manassas, USA) were grown in DMEM (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (10,000 U/ml, Gibco, Gaithersburg, MD, USA). Cells were kept at 37 °C in an atmosphere of 5% CO₂ in 75 cm² tissue culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) and, for all the experimental procedures, harvested using trypsin-EDTA (Sigma, Zwijndrecht, The Netherlands) in their exponentially growing phase. Cells were tested within the last 3 months by microscopic morphology check and growth curve analysis according to the Cell Line Verification Test Recommendations (ATCC-Technical Bulletin No. 8, 2008). Periodic assays were carried out to detect mycoplasma contamination, and the identity of the cells was confirmed by PCR profiling using short tandem repeats (STR).

Human laryngeal carcinoma epithelial cells (HEp-2, ATCC® CCL-23™) were maintained in MEM Eagle's Medium (EMEM, CytoGen AG, Germany) supplemented with 10% fetal calf serum (FCS, Millipore AG, Germany) and 2 mM stable L-glutamine. BHK-21 cells were grown in Dulbecco's minimum essential medium (DMEM; Gibco) supplemented with 5% FCS. Both cell lines were cultivated in 75-cm² tissue culture flasks (Greiner Bio-One, Germany) at 37°C and 5% CO₂.
Human parainfluenza virus type 3 (HPIV3, ATCC® VR-93, kindly provided by Albert Heim, Hannover Medical School) were propagated on HEp-2 cells and the viral titers were determined by plaque assay as described previously (Shibuta et al., 1981 (link)).
The virulent Streptococcus suis (S. suis) serotype 2 strain 10 and the clinical isolate of group B streptococcus (GBS) serotype III strain NEM316 were kindly provided by Hilde Smith (Wageningen Bioveterinary Research, Lelystad, The Netherlands) and Marcus Fulde (Institute for Microbiology and Epizootics, FU Berlin), respectively. For all infection experiments, cryo-conserved bacterial stocks were used and prepared as described previously (Willenborg et al., 2014 (link)).
The open reading frame of MuV-HN (GenBank accession no.: KM519600) was cloned into the expression vector pCG1 and connected with a sequence coding for a FLAG epitope (DYKDDDDK) at the C-terminal end (Krüger et al., 2015 (link)).