Matrigel 1 100

hPSC line WA09 (WiCell 0062, Madison, WI, USA) was used to derive hNP cells (hNP1^TM 00001) as previously described and was obtained from ArunA Biomedical, Inc. (Athens, GA, USA) [29 (link)]. hNP cells were thawed in proliferation medium containing AB2^TM basal medium supplemented with ANS^TM neural supplement (both from ArunA Biomedical Inc.), 2 mM l-glutamine (Gibco, Waltham, MA, USA), 2 U/mL penicillin (Gibco), 2 μg/mL streptomycin (Gibco), 20 ng/mL fibroblast growth factor 2 (FGF2) (R&D Systems Inc. Inc., Minneapolis, MN, USA), and 10 ng/mL leukemia inhibitory factor (LIF) (Millipore, Billerica, MA, USA) and subsequently plated on cell culture dishes coated with Matrigel 1:100 (B&D Biosciences, Bedford, MA, USA). Differentiation of hNP cultures was induced by substituting the proliferation medium with differentiation medium (the proliferation medium lacking FGF2). Cultures were maintained for up to 28 days in vitro (DIV) with the fresh differentiation medium applied every two or three days. All cell cultures were maintained at 37 °C in a humidified incubator with a 5% CO₂ atmosphere.

Vero cells were maintained in DMEM with 5% fetal bovine serum (FBS) at 37 °C, 5% CO₂. C6/36 Ae. albopictus cells (ATCC CRL-1660) were maintained in L-15 Leibovitz Medium with l-glutamine and 10% FBS at 28 °C. Human neurons were made by differentiating hNP1 cells and were obtained from ArunA Biomedical, Inc. [16 (link)]. Neurons were maintained in medium containing AB2^TM basal medium supplemented with ANS^TM neural supplement (both from ArunA Biomedical Inc. Athens, GA, USA), 2 mM l-glutamine (Gibco, ThermoFisher, Waltham, MA, USA), 2 U/mL penicillin (Gibco, ThermoFisher, Waltham, MA, USA), 2 μg/mL streptomycin (Gibco, ThermoFisher, Waltham, MA, USA), and 10 ng/mL leukemia inhibitory factor (LIF) (Millipore, Billerica, MA, USA) and plated on cell culture dishes coated with Matrigel 1:100 (B&D, Franklin Lakes, NJ, USA).