Rpmi 1640 medium

Porcine Reproductive and Respiratory Syndrome virus type 1 (PRRSV-1) strain PRRS-FR-2005-29-24-1 (Finistère strain; genotype 1.1) was propagated on AM cultured in RPMI 1640 medium supplemented with 10% FCS and 1% Streptomycin/Penicillin/Amphotericin (SPA) solution (Eurobio scientific, Les Ulis, France) for 72 h. The supernatant was clarified by centrifugation for 20 min at 600× g and then purified on Amicon Ultra-15 centrifugal Filters (Sigma-Aldrich, Saint-Quentin Fallavier, France) after a 20 min centrifugation at 4000× g and 4 °C. The PRRSV-1 titer using Tissue Culture Infectious Dose (TCID50) assay protocol on AM was 8.7 × 10⁶ TCID50/mL. The Aujeszky’s disease virus (ADV) strain Kojnok propagation was p45to5m4r on Newborn Pig Trachea (NPTr) cells [25 (link)] in DMEM medium supplemented with 10% FCS and 1% of SPA solution (Eurobio scientific). The ADV titer using TCID50 assay protocol on Madin-Darby Canine Kidney cell line (MDCK) was 3.7 × 10⁷ TCID50/mL.

Co-culture was initiated by seeding RA synoviocytes overnight in 96-well plates at a density of 2 × 10⁴ cells/well in RPMI 1640 medium (Eurobio, Courtaboeuf, France) supplemented with 10% human AB serum (Blood Bank Center in Lyon, France), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin (complete RPMI), as previously described (11 ). The next day, PBMC (1 × 10⁵ cells/well) were pre-incubated for 3 h in complete RPMI with or without different treatments and then, without washes, directly seeded at a 5:1 ratio, in the presence of phytohemagglutinin (PHA, 5 μg/ml). After 48 h, supernatants were collected for analysis (13 (link)).

Stromal cells were seeded overnight, and the next day, PBMCs were added at a 5:1 ratio, with or without phytohemagglutinin (PHA, 5 μg/ml), as described [11 (link), 12 (link)]. Treatments, IL-23 (50 ng/ml), anti-IL-23 antibody (1 μg/ml) (R&D Systems, Minneapolis, USA), or control irrelevant antibody (1μg/ml) (Dendritics, Lyon, France) were added to cell cultures, cells alone or co-cultures. After 48 h, supernatants were collected for the analysis and cells for CD3, CD4, CD69, and CD86 staining.
For mRNA expression, co-cultures were initiated by seeding synoviocytes or skin fibroblasts overnight in 12-well plates at a density of 15 × 10⁴ cells/well in a complete RPMI 1640 medium (Eurobio, Courtaboeuf, France, RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 100U/ml penicillin/streptomycin). The next day, PBMCs (75 × 10⁴ cells/well) were seeded in a complete RPMI medium, without or with PHA (5 μg/ml) or IL-23 (50 ng/ml). After 24 h, cells were recovered for RNA extraction.

Alveolar macrophages used for the viral titration were obtained from bronchoalveolar lavage (BAL) of lungs collected from 5 to 7-month-old Large White conventionally bred sows. The animals were bred in accordance with European regulations by the experimental unit of animal physiology of Orfasière (UE PAO), Nouzilly, France. They were serologically tested frequently and known to be free of any common viral infection (swIAV, PRRSV, Porcine Circovirus 2, amongst others). In order to reduce the use of animals, the lungs were collected from pigs slaughtered in the course of the regular management of the experimental unit’s herds. As a consequence, no trial number has been attributed since an experimental authorisation was not requested. Once isolated, the lung airways were infiltrated with 250 mL of phosphate buffered saline (PBS) (Eurobio scientific) supplemented with 2 mM EDTA (Sigma-Aldrich, Saint-Quentin, France). The BAL was then collected, centrifuged, and passed through 40 µm cell strainers. After treatment with erythrocyte lysis buffer (10 mM NaHCO₃, 155 mM NH4Cl, and 10 mM EDTA), AM were washed with PBS, counted and seeded onto sterile plates and flasks for virus titration and propagation. Roswell Park Memorial Institute medium (RPMI) 1640 medium (Eurobio scientific) supplemented with 10% FCS and 2% of SPA solution was used for AM culture.

THP1 cells (ATCC^® TIB-202^TM) were grown at 37 °C and 5% CO₂ in RPMI 1640 medium (Eurobio, Courtaboeuf, France) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies, Saint Aubin, France) and 50 μg/mL Pen-Strep (Invivogen, Toulouse, France). THP1-XBlue cells, which contain a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of NF-κB and AP-1, were acquired from Invivogen and grown at 37 °C and 5% CO₂ in RPMI 1640 medium supplemented with 10% FBS, 100 µg/mL Normocin^TM, and 50 μg/mL Pen-Strep. THP1-XBlue. U937 cells (ATCC^® CRL-1593.2^TM) were grown at 37 °C and 5% CO₂ in RPMI 1640 medium supplemented with 10% FBS and 50 μg/mL Pen-Strep.

The B-ALL cell lines Nalm-6, RS4;11, Reh, HAL-01, and RCH-ACV (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ®, Braunschweig, Deutschland) were cultured in RPMI 1640 medium (Eurobio®, Courtaboeuf, France) containing 10% fetal bovine serum (FBS, Eurobio®), 2 mM of L-glutamine (Eurobio®) with 5000 UI/l penicillin and 50 mg/l streptomycin (Eurobio®). B-ALL cell lines were maintained at 37 °C in a 5% CO2 humidified atmosphere. All cell lines were authenticated via short tandem repeat (STR) methodology and routinely tested for mycoplasma contamination using the MycoAlert PLUS detection kit (Lonza, LT07-705).

4T1 and B16-F10 cells were cultured in RPMI-1640 medium (Eurobio, Toulouse, France) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and cultivated in a humidified incubator at 37 °C in 5% CO₂.