Dulbecco s modified eagle medium

NIH3T3 cells (ATCC, CRL-1658) were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, 11965) supplemented with 10% bovine serum (Thermo Fisher Scientific, 16170-060) and 1% penicillin/streptomycin (Thermo Fisher Scientific, 15140). The cells were maintained at 37 °C and 5% CO₂. HeLa cell line (ATCC and CCL-2) and U2OS human osteosarcoma cell line (Bioresource Collection and Research Center, Hsinchu, Taiwan) were maintained at 37 °C, 5% CO₂ in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 10437) and 1% penicillin/streptomycin (Thermo Fisher Scientific, 15140). Lipofectamine 2000 (Thermo Fisher Scientific, 11668) was used for plasmid transfections based on the manufacturer’s protocol. PES-Cl (Calbiochem, 5.31067) treatments were done by supplementing 50 μM of the drug in cell culture medium 2 h before light-assisted peroxisome damage. Cellular catalase inhibition was done by treating cells with 3-AT (Sigma-Aldrich, A8056) at the concentration of 150 mM for 14 h. Cellular ATM kinase inhibition was done by treating cells with KU-55933 (Sigma-Aldrich, SML1109) at 500 nM 2 h before 559 nm light-assisted peroxisome damage.

HEK293T and HEK293T-hACE2 (Human, female) cells were cultured in a humidified 37°C incubator (5% CO₂) in Dulbecco’s Modified Eagle Medium (GIBCO) supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin, and were passaged when nearing confluency using 1X Trypsin-EDTA.
Calu-3 cells (Human, male), a lung-derived adenocarcinoma cell line, were obtained from Jonas Klingstrom (Karolinska Institutet). Calu-3 cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with Ham’s F-12 (Thermo Fisher Scientific), 2% Fetal Bovine Serum and 1% Penicillin/Streptomycin in a humidified 37°C incubator (5% CO₂) and were passaged using 0.5X Trypsin-EDTA.
Vero E6 cells (ATCC-CRL-1586, African Green Monkey) were maintained in DMEM (GIBCO) supplemented with 2% fetal calf serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO₂ at 37°C.

All mice were breed and housed in a pathogen-free environment and used at 7–14 weeks of age for all experiments. NS1-GFP virus was a generous gift from the Adolfo Garcia-Sastre laboratory. Influenza A viruses PR8 (H1N1) and NS1-GFP [27 (link)] were grown in the allantoic cavity of day 10 chicken embryos as described previously [26 (link)]. Mice were infected with 250 EID₅₀ units of PR8 (0.1LD₅₀), or 10⁵ EID₅₀ NS1-GFP [26 (link)]. All infectious doses were administered i.n. in 50μL of serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) following ketamine and xylazine anesthesia. For i.n. transfer of cells, 500,000 AlvMΦs were given in 50uL of serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) following ketamine and xylazine anesthesia. Irradiation and bone marrow transplantation mice were irradiated with 9.5 Gy and, within 24hours, i.v. injected with RBC-lysed bone marrow cells (1–3 × 10⁶) [26 (link)]. For AlvMΦ depletion CD11c-DTr+ and CD11c-DTr- BALB/C littermates were given 40ng of DTx i.n. following ketamine and xylazine anesthesia. Acivicin was diluted in serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) and 2.5mg/kg was given i.n. in 50uL following ketamine and xylazine anesthesia. 10mg/kg of Zafirlukast was given daily on days 0–3 PI by i.p. injection in 1mL of saline with 1%DMSO and 2% hydroxypropyl-β- cyclodextrin (HPCD).

OC-1 and OC-2 cells [16 (link)] were grown at 33 °C and 5% CO₂ in Modified Eagle Media with Glutamax (Gibco: 41090-036) supplemented with 10% fetal bovine serum and 50 U/ml of γ-Interferon. Neuro-2a (N2A) cells were grown at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum and 1% streptomycin and penicillin. HeLa cells were grown at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

HeLa cells were cultured in Dulbecco’s modified Eagle Medium with 4.5 g/l d-glucose and l-glutamine (Gibco) and A549 cells were cultured in Dulbecco’s modified Eagle Medium/F12 (1:1) with l-glutamine and 15 mM HEPES (Gibco). Both were supplemented with 10% fetal bovine serum (Gibco) and 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco). Cells were maintained in an incubator at 37°C with 5% CO₂.

A line of mouse vascular smooth muscle cells (ATCC, USA) was cultured in high-glucose Dulbecco's modified Eagle medium (Gibco BRL, Grand Island, USA) with 10% foetal bovine serum (Gibco BRL). The mouse macrophage cell line RAW264.7 (ATCC) was also cultured in high-glucose Dulbecco's modified Eagle medium (Gibco BRL) with 10% foetal bovine serum (Gibco BRL). Cells were maintained at 37°C with 5% CO₂ in a humidified environment.