Hepes

The pellet was resuspended in K1 medium [DMEM:HAM’s F12; 1:1 vol/vol; Gibco Life Technologies) supplemented with 25 μg/ml of epithelial growth factor (EGF; Sigma Chemical Co.; St. Louis, MO, USA), 25 μM HEPES (Invitrogen; Carlsbad, CA, USA), hormone mixture (Table 3) and 5% fetal calf serum (FCS; Invitrogen)]. Proximal tubular epithelial cells were obtained by direct culture after removing peritubular endothelial cells.Table 3

Components of hormone mixture in K1 medium

Ingredient	Supplier	Stock solution	Vol./wt. of addition
Insulin	Sigma Chemical Co., St. Louis, MO, USA	Powder	50 mg dissolved in 10 ml of HBSS/HEPES* and small amount of NaOH
Prostaglandin E1	Cayman Chemical, Ann Arbor, MI, USA	0.5 mg/ml (in EtOH)	25 μl
3,3,5-triiodothyro–nine	Sigma Chemical Co.	16.9 mg/ml (0.026 M) (in EtOH)	2 μl of stock added to 10 ml HBSS/HEPES* then used in 100 μl aliquots
Transferrin	Sigma Chemical Co.	Powder	50 mg
Sodium selenite	Sigma Chemical Co.	0.173 mg/ml (10-6 M) in HBSS/HEPES	100 μl
Hydrocortisone	Sigma	0.18 mg/ml (in EtOH)	1 ml

The hormone mixture was brought to a final volume of 100 ml with HBSS/HEPES, and aliquoted into 5 ml or 10 ml portions and stored at –80°C.

*HBSS/HEPES = Hanks Balanced Salt Solution without calcium and without magnesium (Gibco, Life Technologies; Grand Island, NY, USA)/1% HEPES.

Hippocampi from WT and Syt1 KO mice were isolated, collected in ice-cold Hank’s buffered salt solution (HBSS; Sigma) buffered with 1 mM HEPES (Invitrogen), and digested for 20 min with 0.25% trypsin (Invitrogen) at 37 °C. After washing, neurons were dissociated using a fire-polished Pasteur pipette and resuspended in Neurobasal medium supplemented with 2% B-27, 1% HEPES, 0.25% GlutaMAX, and 0.1% Penicillin-Streptomycin (all Invitrogen). Neurons were counted in a Fuchs-Rosenthal chamber and plated at 1.5 K per well in a 12-well plate. Neuronal cultures were maintained in Neurobasal medium supplemented with 2% B-27, 1% HEPES, 0.25% GlutaMAX, and 0.1% Penicillin-Streptomycin (all Invitrogen), at 37 °C in a 5% CO humidified incubator.
Autaptic hippocampal cultures were prepared as described previously^{41 (link)}. Briefly, micro-islands were prepared with a solution containing 0.1 mg/ml poly-D-lysine (sigma), 0.7 mg/ml rat tail collagen (BD Biosciences) and 10 mM acetic acid (Sigma) applied with a custom-made rubber stamp (dot diameter 250 μm). Next, rat astrocytes were plated at 6–8 K per well in pre-warmed DMEM (Invitrogen), supplemented with 10% FCS, 1% Penicillin-Streptomycin and 1% nonessential amino acids (All Gibco).

The spontaneously immortalized human Moorfield/Institute of Ophthalmology-Müller 1 cell line (MIO-M1), initially derived from postmortem human retina, was established, characterized [14 (link)] and demonstrated to express markers of neural progenitors [15 (link)]. MIO-M1 Müller and HEK293 cells were cultured in 1X DMEM/F-12 (1:1) medium containing 2.5 mM L-glutamine, 15 mM HEPES (Thermo Scientific HyClone, Loughborough, UK) and 10% vol/vol fetal bovine serum (filtered, heat inactivated; Gemini Bioproducts, Sacramento, CA) in T175 flasks. Cells were kept in a humidified 5% CO2 incubator at 37°C until collection for RT-qPCR experiments.

Primary 83,320 KPC cells have previously been isolated from end-stage Pdx1-Cre, LSL-Kras^G12D/+, LSL-Trp53^R172H/+ KPC mice (27 (link), 29 (link), 35 (link)). Telomerase-immortalized fibroblasts (TIFs) were also generated previously (18 (link), 28 (link), 76 (link)). KPC cells, TIFs, and human embryonic kidney (HEK) 239T cells were maintained in Dulbecco’s modified Eagle’s media (DMEM; high glucose, pyruvate; Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone) and 10 mM Hepes (Gibco). TKCC05 cells were maintained in media consisting of DMEM/Ham’s F12 media (Gibco) supplemented with 7.5% FBS, 10 mM Hepes, 13.3 mM glucose, hydrocortisone (40 ng/ml), insulin (0.1 IU/ml), and human recombinant epidermal growth factor (EGF; 10 ng/ml). TKCC10 cells were maintained in 1:1 M199 media/Ham’s F12 medium (Gibco) supplemented with 7.5% FBS, 15 mM Hepes, 2 mM glutamine, 1× MEM vitamins, apotransferrin (25 ng/ml), insulin (0.2 IU/ml), 6.5 mM glucose, hydrocortisone (40 ng/ml), EGF (20 ng/ml), triiodothyronine (0.5 pg/ml), and O-phosphoryl ethanolamine (2 μg/ml). All cells were cultured in the presence of penicillin-streptomycin (100 U/ml and 100 mg/ml, respectively) and maintained at 37°C and 5% CO₂. Experiments were conducted at 20% oxygen in a Heracell 150i CO₂/O₂ incubator for KPC, TIF, and TKCC05 cell lines and at 5% oxygen for TKCC10 lines. All cell lines were mycoplasma-free.

Mosquito (Aedes albopictus) C6/36 cells (ATCC CRL-1660) were maintained in minimum essential medium (MEM; Gibco) supplemented with 5% fetal bovine serum (FBS; HyClone), 100 U/mL penicillin and 100 mg/mL penicillin/streptomycin (P/S; Gibco), 0.1 mM nonessential amino acids (NEAA; Gibco), HEPES (Gibco), and 2 mM GlutaMAX (Gibco), followed by incubation in the presence of 5% CO₂ at 32°C. Vero cells (ATCC CCL-81) and VF-Hi and VF-Lo cells (generated from this study) were maintained in Dulbecco modified Eagle medium (DMEM; Gibco) supplemented with 10% FBS, P/S, NEAA, and HEPES and incubate in 5% CO₂ at 37°C. DENV variants were generated by site-directed mutagenesis using Q5 high-fidelity DNA polymerase (NEB), followed by DENV reverse genetics (see below). The Env and prM of all DENV variants were sequence confirmed. DV1, -2, and -3 and 4-WT viruses were grown in C6/36 or Vero cells maintained in infection media. C6/36 infection media contain Opti-MEM (Gibco) supplemented with 2% FBS, 1% P/S, 0.1 mM NEAA, 1% HEPES, and 2 mM GlutaMAX. Vero infection medium is the same as the growth medium except with a 2% FBS supplement.