Dulbecco modified eagle medium (dmem)

Skin organ cultures were prepared from tissue samples derived from abdominal or breast tissues of healthy women (30–40 years old) undergoing routine therapeutic procedures, with the donor’s consent. Samples were collected by ALPHENYX (Marseille, France). ALPHENYX possesses the required accreditations from the French Ministry of High Education and Research, in addition to the Committee of Persons Protection under the authorization numbers AC-2014-2141 and DC-2014-2147.
Microdissected human skin was cultured at 37 °C under 5% CO₂, in DMEM (Dutscher, Brumath, France, #L0060-500) plus 10% FBS (Sigma Aldrich, #F7524), 1% Penicillin/Streptomycin (Sigma Aldrich, St. Louis, MO, USA, #P0781-100), and 1% of minimum medium non-essential amino acids 10X (ThermoFisher, Waltham, MA, USA), #11140-035).
NHEKs cells were isolated from human skin epidermis and cultured in KGM2 Medium (Promocell, Heidelberg, Germany), #C-20011) with 1% Penicillin/Streptomycin (Sigma Aldrich, #P0781-100) at 37 °C under 5% CO₂ and 95% relative humidity. The detailed protocols are described in the supplementary materials.

To study S. aureus interaction with endothelial cells we used the EA.hy926 cell line (ATCC® CRL-2922™), originally derived from human umbilical vein. EA.hy926 cells were grown in Dulbecco's modified Eagle high glucose medium (DMEM, Dominique Dutscher™) supplemented with 10% fetal bovine serum (FBS, Eurobio™). Culture media contained 1% penicillin/streptomycin and 1% amphotericin B. Antibiotics were removed from culture medium prior to infection.
The human monocytic cell line THP-1 (ATCC® TIB-202™) was grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 5% FBS. Two days before infection, phorbol myristate acetate 200 ng mL⁻¹ (PMA) was added to the culture medium to induce monocytes differentiation into macrophages.
All cells were incubated in a humidified 5% CO2 atmosphere at 37°C.

The influenza D virus strain D/bovine/France/5920/2014 was isolated from the lung of a dead calf with clinical signs of BRD (9 ). The virus was propagated on human rectal tumor cells (ATCC CRL-11663) in Dulbecco’s modified Eagle’s medium (DMEM; Dutscher, France) supplemented with 1 μg/ml TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (Thermo Fisher Scientific, MA, USA) at 37°C, 5% CO₂ for 5 days. Viral titer was determined on swine testis cells (ATCC CRL-1746) using the 50% tissue culture infective dose (TCID₅₀) method as previously described (17 (link)).
M. bovis strain RM16 was isolated in 2016 in France from a pool of transtracheal aspiration samples from 3 heifers with clinical signs of respiratory infection (48 (link)). Mycoplasma cells were grown in SP4 medium (49 ) supplemented with cephalexin (500 μg/ml). After 24 to 36 h of incubation at 37°C, mycoplasma cultures were stored at −80°C. Mycoplasma titers were determined by serial dilutions in Dulbecco’s phosphate-buffered saline (PBS; Invitrogen) supplemented with 1% heat-inactivated horse serum (Invitrogen). Dilutions were spotted (10 μl) onto solid SP4 medium, and CFU were counted after 2 to 5 days of incubation at 37°C. For animal inoculations, mycoplasma cells were washed twice in DMEM by 20 min of centrifugation at 9,000 × g and kept on ice. Prior to inoculation, 10¹⁰ CFU were diluted in 10 ml DMEM.