Basal medium eagle bme

Cells of the Vero (African green monkey kidney) and BHK-21 (baby hamster kidney) lines were maintained at 37 °C in Dulbecco’s modified eagle serum (DMEM; Sigma-Aldrich, St. Louis, MO, USA) media containing 10% heat-inactivated foetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA, in a humidified incubator supplemented with 5% CO₂ atmosphere, whereas C6/36 (Aedes albopictus) cells were maintained at 28 °C in Basal Medium Eagle (BME; Sigma-Aldrich, St. Louis, MO, USA) media supplemented with 10% heat-inactivated FBS [13 (link),14 (link)]. Viral stocks of DENV-2 (New Guinea C; M29095) were propagated in C6/36 cells [14 (link)], and of ZIKV (Asian/Cook Islands/2014) and WNV (MRM61C) in Vero cells; cells at 80% confluency were infected at a multiplicity of infection (MOI) of 0.1. At 48 h, when >70% of the cells were detached, the supernatant was harvested as the virus stock. Viral titre was subsequently determined by plaque assay (see below).

Astrocyte culture media comprised Basal Medium Eagle (BME, Sigma-Aldrich, Milwaukee, WI, USA) and Ham’s F-12K media with L-glutamine (ATCC, Manassas, VA, USA), 5% horse serum, 5% fetal bovine serum, and penicillin-streptomycin (Sigma-Aldrich, Milwaukee, WI, USA) as previously described by Daniel and DeCoster [18 (link)]. Then, the mixture was filtered using a sterile filtration unit (Thermo Fisher Scientific, Waltham, MA, USA).

To evaluate the cellular anchorage-independent growth, the soft agar colony formation assay was conducted according to modified protocol [26 (link)]. A mouse epithelial JB6 Cl41 cell was obtained from the ATCC (Manassas, VA, USA) and cultured in Eagle’s minimum essential medium (MEM; GIBCO, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 5% fetal bovine serum (FBS; GIBCO), 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO). Basal Medium Eagle (BME; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 2 mmol/L L-glutamine, and 25 μg/mL gentamicin was mixed with 0.6% agar containing DMSO, EGF (10 ng/mL) or TPA (10 ng/mL), and/or each compound (50 μM) and solidified as the bottom agar of 6-well plates. JB6 Cl41 cells (8000 cells/well) were suspended in 1 mL of BME medium supplemented with 0.3% agar containing DMSO, EGF (10 ng/mL) or TPA (10 ng/mL) and/or each compound (50 μM) and added to the bottom agar layer. The plates were incubated (37 °C) in a 5% CO₂ incubator for two weeks. Colonies were visualized by a microscope (Leica Microsystems, Germany) and the numbers analyzed using an Image-Pro Plus software ver.6.1 (Media Cybernetics, Rockville, MD, USA).

HEK293FT cells (Thermo Scientific) were maintained in Dulbecco modified eagle media (DMEM) containing 4.5 g L⁻¹ glucose, 2 mM l-glutamine, and supplemented with 10% fetal bovine serum and 1% Pen Strep antibiotic (Thermo). Cells were maintained at 37 °C in 5% CO₂. All procedures with animals followed the guidelines approved by the Institutional Animal Care and Use Committee of National Institute on Aging. Primary neuronal cultures were prepared from newborn P0 C57Bl/6 J mice. Dissected hippocampi were incubated in 10 mL Basal medium eagle (BME) (Sigma) supplemented with 5 mL papain solution (Worthington) for 30 min at 37 °C. Brains were then incubated with 5 μg of DNAseI and titurated to dissociate single cells. Cells were washed with two cycles of 10 mL BME and counted. Cells were plated at ~0.5 × 10⁶ on Poly-d-lysine & laminine precoated coverslips in BME supplemented with B27, N2, 1 mM Glutamax (Invitrogen), 0.45% glucose (SIGMA). Media was replaced the next day with BME supplemented with 2.5 μM cytosine arabinose to kill off glial cells.

Primary cortical neuronal and astrocyte cultures were prepared from C57BL/6J newborn (P0) pups. First, dissected mouse cortices were incubated in 1 ml/cortex Basal Medium Eagle (BME) (Sigma-Aldrich), containing 5 U of papain/ (Worthington) for 30 min at 37°C. Five μg of DNase I was added to each cortex preparation, and brain tissue was dissociated into single cells. Cells were washed twice with 10 volumes of BME and counted. Neuronal cultures were plated at 0.6×10⁶ cells/cm² in BME supplemented with B27, N2, 1 mM glutaMAX-I (all from Invitrogen), 0.45% glucose (Sigma-Aldrich), and 2.5 μM cytosine arabinoside (Sigma-Aldrich) for inhibition of glial cell proliferation. Astrocyte cultures were plated at 0.3×10⁶ cells/cm² in DMEM media (Thermo Scientific), supplemented with 10% FBS (Lonza) into tissue culture flasks. For the preparation of purified astrocyte cultures, 7–10 day primary cultures were vigorously shaken to detach microglia and oligodendrocytes. Purity of each culture was assessed with MAP2 for neurons, GFAP for astrocytes, and OLIG2 and IBA1 to exclude oligodendrocytes and microglia. Cultures had 70%–90% of neurons or astrocytes in all experiments.