Amphotericin b solution

Cortices from 1-day-old pups were extracted and placed onto a 100-mm petri dish using aseptic techniques. Cortices were sliced with a commercial razor blade, further broken up with a rigorous up-and-down motion in 10 mL of medium, and filtered with a 70-μm filter. The cells were then plated onto a 100-mm petri dish and put in an incubator of 37 °C with 5 % CO₂. Cell culture medium DMEM/F12 (Wisent, St. Bruno, QC) was supplemented with 10 % FBS (Invitrogen, Burlington, ON), 1 % penicillin-streptomycin solution (Wisent, St-Bruno, QC), 1 % L-glutamine solution (Wisent, St. Bruno, QC), 0.9 % sodium pyruvate solution (Wisent, St. Bruno, QC), 0.9 % MEM amino acid solution (Wisent, St. Bruno, QC), and 0.9 % amphotericin B solution (Wisent, St. Bruno, QC). The medium of the mixed glial culture was changed every 2 to 3 days. After 3 weeks, primary microglia cells were separated from astrocytes using EasySep CD11b positive selection kit following the manufacturer’s instructions (Stem cell, Vancouver, BC).

Brains were extracted and meninges removed from 1-day-old pups. Tissue was chopped and passed through a 70-μm filter. The cells were cultured in DMEM/F12 medium with 10% FBS (Invitrogen, Burlington, Canada) supplemented by 1% penicillin-streptomycin solution, 1% L-glutamine solution, 0.9% sodium pyruvate solution, 0.9% MEM amino acid solution, and 0.9% amphotericin B solution (all from Wisent, St. Bruno, Canada). The medium of the mixed glial culture was changed every 2 to 3 days. Primary glial cells were ready for experiments after 3 weeks. Glial cells were treated with LPS (100 ng/mL) and/or IFNγ (10 ng/mL) for 24 hours, and the conditioned media were collected and stored at −80°C.

Newborn pig tracheal epithelial cell line (NPTr) and immortalized porcine alveolar macrophage (iPAM 3D4/21) cell line were used for all PCV2b and SwIV single and co-infections. Madin–Darby Canine Kidney (MDCK) cell line was used for SwIV titration and propagation. The NPTr cell line was kindly provided by Dr. M. Ferrari (Instituto Zooprofilattico Sperimental, Brescia, Italy) [48 (link)]. The NPTr and the MDCK (ATCC CCL-34) cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada) supplemented with 10% fetal bovine serum (FBS) (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada), 1 mM sodium pyruvate, 10 I.U./mL of penicillin, 10 μg/mL of streptomycin and 250 g/L amphotericin B solution (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada) [48 (link)]. The iPAM 3D4/21 cell line (ATCC CRL-2843) was maintained in RPMI 1640 medium (Invitrogen Corporation, GibcoBRL, Burlington, ON, Canada) with 10% FBS and adjusted to contain 2 mM L-glutamine (Invitrogen), 10 mM HEPES (Invitrogen), 1 mM sodium pyruvate, 1 mM non-essential amino acids (Invitrogen) and 0.1 mg/mL streptomycin/100U penicillin solution (Invitrogen). All cells were cultivated in a humidified incubator in 5% CO₂ atmosphere at 37 °C.