F 12k medium

MCF-10A cells (ATCC CRL-10317) were cultured in MEBM medium,
supplemented with 0.1% insulin, 0.1% hEGF, 0.4% hydrocortisone, and 10% cholera
toxin. MCF-7 cells (ATCC HTB-22) were cultured in DMEM (Fisher Scientific,
BW12719F), supplemented with 10% fetal bovine serum (FBS), 0.1 mM MEM
Non-Essential Amino Acids (NEAA), 2 mM L-glutamine, and 1% Pen-Strep. BEAS-2B
cells (ATCC CRL-9609) were cultured in BEGM BulletKit (Lonza, CC-3170). A549 cells
(ATCC CRM-CCL-185) were cultured in F-12K medium (Fisher Scientific, MT10025CV),
supplemented with 10% FBS and 1% of Pen-Strep. Jurkat cells (ATCC TIB-152) were
cultured in RPMI 1640 medium (Thermo Fisher Scientific, Grand Island, NY USA),
supplemented with 10% fetal bovine serum (FBS), and 1% Pen-Strep. All cell
cultures were maintained at 37 °C in 5% CO₂ and routinely
passaged, per published protocols^{42 ,43} , once they reached 80% confluence.
Cells were dissociated by treatment with 0.25% trypsin/EDTA for
either 3 min (MCF-7 and A549 cells) or 5 min (MCF-10A and BEAS-2B cells) at 37 °C
(Refs. 44 (link)–46 ), washed with the respective growth media,
centrifuged at 0.2 RCF, and re-suspended at a concentration of ~20 000 cells per
mL in PBS. To ensure cell viability, cells were injected into the prepared devices
for screening immediately following re-suspension.

Clone 9 hepatocyte, RFL-6 and RMC cells were obtained from American Type Culture Collections (ATCC, Manassas, VA). Upon receipt, cells were passaged 3 times to create cryopreserved stocks. All cell lines were grown per ATCC recommended culture conditions. Specific media for each cell line are as follows: F-12 K medium (Fisher Scientific, Florence, KY) with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) for Clone 9, F-12 K medium with 20% FBS for RFL-6, and DMEM (ATCC) with 15% FBS for RMC. Cryopreservation medium was complete growth medium supplemented with 5% (v/v) DMSO. All cells were grown at 37°C, 5% CO₂. Cells were subcultured every 48–72 h, when cell density reached 75–90%.
All cell lines were authenticated for correct species and verified free of interspecies or mycoplasma contamination by IDEXX (Westbrook, ME).

Human immortalized uroepithelial cells (SV‐HUC‐1) and 293T cells (ECACC Cat# 12022001) were purchased from the American Type Culture Collection (Manassas, VA, USA), while human BC cell lines (5637, J82, T24, and UM‐UC‐3) were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human BC cell lines T24 and 5637 were maintained in RPMI‐1640 medium (Gibco, Cat. #: C11875500BT). J82, UM‐UC‐3, and HEK‐293 T cells were grown in DMEM (Gibco, Cat. #: C11885500BT). SV‐HUC‐1 cells were cultured in F‐12K medium (Fisher Scientific, Cat. #: 21127‐022). All culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, Cat. #: 10270106) and 100 U/ml penicillin/streptomycin (Life Technologies, Cat. #: 15140‐122). All cells were cultured at in a 37°C humidified incubator with 5% CO₂. All cell lines were free of mycoplasma infection.

Murine macrophage RAW264.7 cells (ATCC®, Chicago, USA) were incubated in Dulbecco's Modified Eagle's Medium (DMEM; Fisher) containing 10% (v/v) fetal bovine serum (FBS; BI, Israel) and 1% (v/v) penicillin-streptomycin (Gibco™, USA). The human prostate cancer cell lines PC-3 and DU145 (ATCC®, Chicago, USA) were maintained in 50% DMEM and 50% F-12K medium (Fisher) supplemented with 10% FBS and 1% penicillin-streptomycin. These cell lines were incubated at 37°C in a 5% CO₂ humidified incubator. PMF samples used for cell assays were prepared as 1,000 × stock solutions in DMSO and unless otherwise stated, were maintained at 20°C until used.

Human A549 lung epithelial cells ATCC^® CCL-185^™ (American Type Culture Collection, Manassas, VA) were cultured in F-12K Medium (Life technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Life technologies) with or without 1% 100× antibiotic-antimycotic (Life technologies). Cell suspensions were either plated onto 12- or 24-well tissue culture plates (BD Falcon, Bedford, MA) or on 8-well culture chambers (BD Falcon) depending on the specific experiment. All cell cultures were incubated at 37°C in a 5% CO₂ humidified atmosphere.

HEK293T cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), human lung carcinoma cells (A549) were cultured in F12K medium (Life Technologies) supplemented with 10% FBS, and Madin-Darby canine kidney (MDCK) cells were cultured in MEM (Life Technologies) containing 5% newborn calf serum (Sigma-Aldrich). All cells were maintained in a humidified incubator containing 5% CO₂ at 37°C.
A/chicken/Vietnam-Ca Mau/1180/2006 (VN/1180, H5N1) and A/Anhui/1/2013 (AH/1, H7N9) were grown in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs, as reported previously [6 (link),42 (link)]. A/WSN/33 (WSN, H1N1) and a 2009 pandemic H1N1 virus, A/Fuzhou/1/2009 (FZ/1, H1N1), were propagated in MDCK cells cultured in MEM containing 0.3% bovine serum albumin (BSA, Sigma-Aldrich) and 0.5 μg/ml N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich).