Roswell park memorial institute (rpmi)

Tohoku Hospital Pediatrics-1 (THP-1, male, 1 year old) cells were cultured in RPMI ([RPMI]; Wisent, Montreal, QC, Canada; Cat#: 350-007-CL) containing 10% fetal bovine serum (ScienCell, Carlsbad, CA, USA, Cat#: 0010) and maintained at 37°C in a 5% CO2 humidified incubator. Cells were maintained in T175 Nunc EasYFlask Cell Culture Flasks (ThermoFisher, Waltham, MA, USA, Cat#: 159910) at a cell density between 1x10⁵ cells/ml and 1x10⁶ cells/ml (passages 10–12 were used). Source cell vials were monitored and subsequently tested negative for the presence of mycoplasma contamination (Lonza, Gampel, GB, Switzerland, Cat#: LT07-118). STR profiling for cell line authentication (GenePrint 10 System, Promega) was performed by The Center for Applied Genomics (TCAG), The Hospital for Sick Children, Toronto, Canada (Table S2). DNA was isolated from cells using the PureLink Genomic DNA Mini Kit (Invitrogen, Cat #K182001) according to the manufacturers protocol, quantified via Nanodrop, and diluted to a concentration of 30 ng/μL for profiling.

One million PBMC from uninfected donors were stimulated for 18 h in 1 mL complete medium [RPMI (Wisent) + 10% FBS (Wisent) + 1% Penicillin/Streptomycin (ThermoFisher)] with 1 μg/ml of LPS (Sigma), βDG (Sigma) or both. Supernatants were collected and CXCL13 was measured as previously described.

Remaining cytospin BAL cells were pooled with the three additional 1 ml lavages, then centrifuged at 800 g 4°C, re-suspended in RPMI (Wisent Bioproducts, St-Bruno, QC, Canada) and macrophages were isolated by adherence to a culture-treated 96-well plate (5.0 × 10⁴ pulmonary macrophages per well; 1 h at 37°C, 5% CO₂). Cells were washed with PBS and lysed immediately for RNA isolation using the RNeasy Mini Kit (QIAGEN, Toronto, ON, Canada) containing 1% β-mercaptoethanol (Millipore Sigma, Oakville, ON, Canada) following manufacturer’s instructions. Lysates were kept at −80°C until RNA extraction. Separate wells of macrophages were washed with PBS and cultured with RPMI for 24 h at 37°C, 5% CO₂. Supernatants were then collected and stored at −80°C for ELISA analysis.

The human hepatocarcinoma Huh7 cell line was kindly provided by Hugo Soudeyns. Murine LL171 reporter cells (71 (link)), Crandell-Rees Feline Kidney (CRFK), and Huh7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution (all from Wisent, Inc., Canada). Hepatocarcinoma RIG-I-deficient Huh7.5 (a kind gift from Patrick Labonté), and Vero E6 (kindly shared by Tom Hobman) cells were cultured in DMEM (Life Technologies) containing 10% FBS, 1% PS, and 1% nonessential amino acids (Thermo Fisher). Human embryonic kidney HEK293T and human monocytic cell line THP-1 were grown in RPMI (Wisent) with 10% FBS and 1% PS. Frozen human peripheral blood mononuclear cells (PBMCs) were obtained using Institutional Review Board (IRB)-approved consent forms and protocols (Stem Cell Technologies). They were thawed in 20% FBS containing RPMI prewarmed at 37°C, washed twice, and incubated for 6 h. Medium was replaced by RPMI with 10% FBS and 1% PS before cytotoxic and antiviral assays. All cells were maintained at 37°C and 5% CO₂.

Human serous carcinoma-derived ES-2 ovarian cancer cells, HT-29 colon adenocarcinoma, and triple-negative breast cancer cell lines, including MDA-MB-231, MDA-MB-468, MDA-MB-157, BT-20 and HCC-70, were all purchased from the American Type Culture Collection (ATCC). BT-20, ES-2 and HT-29 cells were grown as monolayers in McCoy’s 5a Modified Medium (Wisent, 317-010-CL) containing 10% fetal bovine serum (Life Technologies, 12483-020), 100 U/mL penicillin and 100 mg/mL streptomycin (Wisent, 450-202-EL). MDA-MB-231, MDA-MB-157 and MDA-MB-468 breast cancer cell lines were grown in DMEM Medium (Wisent, 319-005-CL) supplemented with 10% of fetal bovine serum, while HCC-70 cells were cultured in RPMI (Wisent, 350-007-CL). All cells were cultured at 37 °C under a humidified 95%–5% (v/v) mixture of air and CO₂.

HEK293T and Vero cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Wisent Bioproducts, Saint-Bruno, QC, Canada) and Minimum Essential Medium (MEM, Sigma-Aldrich, St. Louis, MO, USA) respectively, and supplemented with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich), 0.3 mg/mL L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Wisent). Cells were maintained at 37 °C in 5% CO₂ at 100% relative humidity. Bone marrow-derived macrophages (BMDMs) were isolated and differentiated as described previously [26 (link)]. Briefly, mice were euthanized, musculature and connective tissue removed from the tibia and femur, and the bones cut at each end. The bones were placed in a tube containing a hole at the bottom, centrifuged, bone marrow cells re-suspended in DMEM supplemented with 10% FBS (Wisent), penicillin and streptomycin (Hyclone, GE Healthcare, Chicago, IL, USA), and were filtered through a 40 µm filter. Cells were differentiated into macrophages in 20% L929-conditioned media and plated for 7 days. On day 8, cells were seeded into 48-well plates and were placed in Roswell Park MEMorial Institute medium (RPMI, Wisent), supplemented with 10% FBS (Sigma-Aldrich), 0.3 mg/mL L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. All animal procedures were approved by the University of Ottawa Animal Care Committee.