Dulbecco modified eagle medium (dmem)

Bone marrow-derived macrophages (BMMs) were prepared as described previously (Ichinohe et al., 2009 (link)). In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque). Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum (FBS), and L-glutamine (2 mM) at 37°C/5% CO₂. HEK293FT cells (a human embryonic kidney cell line) and HeLa cells (a human epithelial carcinoma cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Nacalai Tesque). MDCK cells (Madin-Darby canine kidney cells) and HT-1080 cells (a human fibrosarcoma cell line) were grown in Eagle’s minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Nacalai Tesque).
Influenza A virus strain A/PR8 (H1N1) was grown at 35°C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs (Ichinohe et al., 2009 (link)). The viral titer was quantified in a standard plaque assay using MDCK cells (Pang et al., 2013 (link)).

The African green monkey kidney cell line, Vero cells (ATCC®CCL-81™), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Vero cells expressing TMPRSS2, VeroE6/TMPRSS2 cells (VeroE6/TMPRSS2, JCRB number; JCRB1819), were maintained in DMEM containing 5% FBS and 1 mg/mL G418 disulfate aqueous solution (Nacalai Tesque) at 37 ℃. IEC#17 cells were differentiated from a human iPSC line, TkDN4-M, as described previously^{33 (link)}. IEC#20, IEC#25, and IEC#29 were also differentiated from the human iPSC lines, 1231A3, 1383D6^{36 (link)}, and TkPP7^{37 (link)}, respectively. To prepare monolayers, IECs were cultured in a multiwell plate at a density of 6.3 × 10⁵/cm² in a culture medium for 2 days. For differentiation, cells were cultured in a differentiation medium for 4 days (for details, refer to Ref.^{38 (link)}). After 6 days, an average of 1 × 10⁵ differentiated IECs were seeded in 96-well plates.

3T3-L1 adipocytes were cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS until two days post-confluence. Then (day 0), the cells were treated with or without adipogenic mixture containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai), 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO), and 1 μM insulin (Nacalai) for 48 h. After 48 h (day 2), medium was replaced with DMEM supplemented with 10% FBS. Until day 7, the cells were maintained in DMEM with 10% FBS.

High-speed live imaging of ciliary beating and ependymal flow assay were performed as described previously with minor modifications^{41 (link),42 (link)}. For the high-speed imaging of ciliary beating, the wholemount preparations of the lateral walls of the LVs were incubated with FITC-labelled rat anti-CD24 antibody (BD Biosciences) in DMEM (Nacalai) for 30 min at RT, rinsed with DMEM, placed on a dissection dish, and fixed with staples. Ciliary beating was recorded with a 10 ms exposure time at 100 frames per second (fps) at RT using an Olympus BX53 microscope, LUMFLN60XW water immersion objective lens (NA 1.10), ORCA-Flash4.0 V3 high-speed camera (HAMAMATSU) and high-speed recording (HSR) software (HAMAMATSU).
For the ependymal flow assay, a glass micropipette filled with fluorescent polystyrene latex microbeads (2 µm, Polysciences) attached to an MO-10 micromanipulator (Narishige) was lowered onto the wholemount, and the microbeads were deposited onto the ventricular surface. The movement of microbeads was recorded at RT at 20 fps using an Olympus SZX16 fluorescent dissection microscope, ORCA-Flash4.0 V3 high-speed camera and HSR software. The speeds of the migrating fluorescent beads were quantified using the Manual Tracking plugin for ImageJ software (written by Dr. Fabrice P. Cordelieres).

African green monkey kidney cells (Vero) (ATCC CCL-81), human kidney cell line 293T (ATCC CRL-3216), and human lung cell line A549 (ATCC CCL-185) were cultured using the Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 5% or 10% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Nacalai Tesque). VeroE6-TMPRSS2 cells temporally expressing TMPRSS2 were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB1819) and cultured with DMEM containing 5% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 mg/mL G418 (Nacalai Tesque). The HEK293-3P6C33 cells, which can exogenously express ACE2 and TMPRSS2 on adding tetracycline, were cultured in DMEM containing 10% FBS and 10 mg/mL blasticidin (InvivoGen, CA, USA). The culture medium was replaced with a fresh medium containing 1 mg/mL doxycycline hydrochloride (Sigma-Aldrich, MO, USA) to express ACE2 and TMPRSS2 in HEK293-3P6C33 cells (36 (link)).