Hepes

Human skin, bladder, and vaginal mucosa biopsies were obtained from patients undergoing plastic surgeries or benign pathologies. Three biopsies of each tissue were obtained, and fibroblasts and epithelial cells were isolated. The biopsy was rinsed, cut into small strips, and incubated overnight at 4 °C in a 500 mg/mL thermolysin solution (Sigma-Aldrich, Oakville, ON, Canada) diluted in a 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES, MP Biomedicals, Montreal, QC, Canada) buffer with 1 mM of CaCl₂ pH 7.4 (Sigma-Aldrich). The epithelium was gently separated from the stroma with forceps. The stroma was incubated for 3 h in a 125 U/mL collagenase H solution (Boehringer Mannheim, Laval, QC, Canada) diluted in Dulbecco-Vogt modification of Eagle’s medium (DMEM, Invitrogen, Burlington, ON, Canada), supplemented with 10% fetal bovine serum (FBS, Avantor Seradigm, Radnor, PA, USA), 100 U/mL penicillin (Sigma-Aldrich, Oakville, ON, Canada), and 25 µg/mL gentamicin (Schering, Pointe-Claire, QC, Canada) at 37 °C. Fibroblasts (DF1, DF2, and DF3 from skin dermis; BF1, BF2, and BF3 from bladder lamina propria; and VF1, VF2, and VF3 from vagina) were grown in DMEM with appropriate serum (depending on the condition) and antibiotics. They were used between passages 3 to 6.

4×10⁵ responding splenocytes from congenic CD45.1 mice were stimulated in vitro with 10μg/ml α-CD3 (145-2C11) (BioXcell) for four days in in RPMI 1640 media (Biochrome, Berlin, Germany) supplemented with 10% FCS (Linaris, Dossenheim, Germany), PenStrep (100U Penicillin, 100μg Streptomycin/ml; Sigma), 10mM Hepes (MP Biomedicals), 1mM Sodium Pyruvat (Sigma), 1x non-essential amino acids (Sigma) and 10μM β-Mercaptoethanol (Sigma). 4×10⁵in vitro activated or IL-2 complex (5μg IL-2 / 25μg α-IL-2) expanded Tregs from CD45.2 wildtype mice were added to selected wells. The proliferation of responding (CD45.1) CD4 and CD8 T cells was measured based on their expression of Ki67.

PCCDS and PCGS were prepared using the Krumdieck tissue slicer. The Krumdieck tissue slicer was filled with ice-cold Krebs-Henseleit buffer with a pH of 7.4, which was supplemented with 25 mM D-glucose (Merck), 25 mM NaHCO₃ (Merck) and 10 mM HEPES (MP Biomedicals, Aurora, USA) and saturated with carbogen (5% O₂/5% CO₂). The arm speed was set at the lowest speed, whilst the blade speed was set at the highest speed. The wet weight of the slices was measured at the start of the slicing and the slicer was adjusted accordingly. The wet weight is generally used as an indicator of thickness of the slices. The wet weight of PCGS was 5–6 mg. As the appearance and diameter of the cystic duct specimens varies between patients, the wet weight of the PCGS was used to adjust the slicer for the PCCDS. The slicer was set to a wet weight of 10 mg for the PCGS and this setting was then used to prepare the PCCDS. At this setting, it was possible to prepare intact PCCDS. Slices were collected and stored on ice in UW solution until transfer into culture plates. Three slices were immediately preserved in 4% formalin.

Human embryonic kidney 293 (HEK293) and human hepatoma Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with HEPES (MP Biomedicals).
Immortalized RIG-I^+/+/MDA5^+/+(wild type), RIG-I^-/-/MDA5^+/+, RIG-I^+/+/MDA5^-/- and RIG-I^-/-/MDA5^-/- mouse embryonic fibroblast (MEF) cell lines were maintained as described previously (18 (link)). Cell media was supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest), penicillin/streptomycin and L-glutamine. Plasmids were transfected into HEK293 and Huh7 cells with TransIT-LT1 Reagent according to manufacturer’s instructions (Mirus Bio LCC, Madison). PolyI:C stimulation was carried out by first transfecting plasmids with TransIT-LT1 Reagent followed by transfection with stimulatory low molecular weight polyI:C (10μg/well in 12-well plates; LMW polyI:C; InvivoGen) with Lipofectamine2000 (Invitrogen). Ebolavirus ssRNA mimic was transfected into MEF cells using Lipofectamine3000 transfection reagent (Invitrogen) (19 (link)).
Spodoptera frugiperda (Sf9) (ATCC: CRL-1711) cells were used for baculovirus expression and they were maintained in TNM-FH medium (Sigma-Aldrich Co.) supplemented with 0.6 µg/ml penicillin, 60 µg/ml streptomycin, 2.5 µg/ml amphotericin B and 10% fetal calf serum (Sigma-Aldrich Co.) or in EX-C420 Serum-Free Medium (Sigma-Aldrich Co.) as recommend by the manufacturer.

VERO C1008 (Vero E6) cells (ATCC CRL-1586™) were maintained in Dulbecco’s Modified Eagle Medium, high glucose, pyruvate, L-glutamine (Gibco) supplemented with 10% foetal bovine serum (CellSera), 10 mM HEPES (MP Biomedicals), and 1 × penicillin–streptomycin (Gibco) at 37 °C and 5% CO₂.
BHK-21 cells (ATCC CCL-10™) were maintained in Minimum Essential Media supplemented with 10% foetal bovine serum, 10% tryptose phosphate broth, 10 mM HEPES, 2 mM glutamine (Gibco), and 1 × penicillin–streptomycin at 37 °C and 5% CO₂.

Murine PCKS were prepared as described in detail by Poosti et al. (2015 (link)). In short, murine kidneys were prepared by removing adipose tissue and slices were made in ice-cold Krebs-Henseleit buffer supplemented with 25 mM D-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO₃ (Merck), 10 mM HEPES (MP Biomedicals, Aurora, OH, USA), and saturated with carbogen (95% O₂, 5% CO₂) using a Krumdieck tissue slicer (Figure 1). The obtained slices were 4.5 mm in diameter and had a wet weight of 4–6 mg, corresponding to an estimated thickness of 250–300 μm. Slices were incubated, up to 96 h, in Williams' Medium E with GlutaMAX (Life Technologies, Carlsbad) supplemented with 10 μg/mL ciprofloxacin and 2.7 g/L D-(+)-Glucose solution (Sigma-Aldrich, Saint Louis) at 37°C in an 80% O₂ and 5% CO₂ atmosphere while gently shaken. Medium was refreshed every 24 h. Murine PCKS were also treated for 48 h with 2.5 μM LY2109761, a TGF-β receptor inhibitor. After incubation, mPCKS were snap-frozen in liquid nitrogen and stored at −80°C until use.