Dulbecco s modified

The titers of SARS‐CoV‐2 used in this study were determined either by plaque assay or by 50% tissue culture infectious dose (TCID₅₀). For the plaque assay, monolayers of VeroE6/TMPRSS2 cells grown in a 96‐well plate were infected with serially diluted culture supernatants of SARS‐CoV‐2, cultured in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich) containing 2.5% carboxymethyl cellulose at 37°C under 5% CO₂ for 3 days and then fixed with 4% paraformaldehyde and stained with crystal violet. Emergent plaques were counted using an optical microscope. For TCID₅₀, 10‐fold serially diluted viruses were mixed with VeroE6/TMPRSS2 cells (2–3 × 10⁴ cells/well) in a 96‐well plate and incubated at 37°C under 5% CO₂ for 5 days. Five days later, the cytopathic effect in each well was checked, and the TCID₅₀ was determined by the Kärber method.¹¹

HEK293T cells were cultured at passage 4–20 in high-glucose Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% v/v fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) without antibiotics and incubated at 37 °C temperature and 5% CO₂ conditions as previously described [44 (link)]. For APP695 with Swedish mutation-overexpressing HEK293 cells, the cultured medium was supplemented with 150 µg/mL of G418 (ThermoFisher Scientific, Waltham, MA, USA). For shedding assay and Western blot experiments, 3 × 10⁵ cells were plated into 6-well plates. DNA plasmid vectors were transfected at 60–90% cell confluency with a DNA-PEI max mixture consisting of 2.5 µg total DNA along with 7.5 µg pH 7.3, polyethylenimine HCl max solution (PEI max, Polysciences, Warrington, PA, USA) in Opti-MEM Reduced-Serum Medium (ThermoFisher Scientific, Waltham, MA, USA).

Experiments were carried out using the mouse 661W cone photoreceptor-derived cell line, generously provided by Dr Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma, Health Sciences Centre, Oklahoma City, OK, USA). This cell line was previously validated by this group [11 (link)] through real-time quantitative polymerase chain reaction (rt-qPCR) analysis for cone specific opsins; blue cone opsin (Opn1sw) and red/green opsin (Opn1mw). Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (FCS) and 1% penicillin streptomycin (PS) and maintained at 37°C in a humidified 5% CO₂ atmosphere. To analyze the effects of Norgestrel (Sigma) on 661W cells, cells were seeded in to the appropriate culture vessel and allowed to attach overnight. Cells were then washed three times with warmed 1xPBS and complete or serum-free medium supplemented with 20 μM Norgestrel or DMSO vehicle (Sigma) added. After incubation for the indicated times, cells were detached using accutase (Sigma) and, together with their supernatant, centrifuged and washed with 1x PBS to leave a whole cell pellet.

Human microglia C20 cells [28 (link)] were grown in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (Sigma Aldrich, Taufkirchen, Munich, Germany), supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 10,000 U/mL penicillin-streptomycin at 37 °C in humidified air with 5% CO₂, and the medium was changed three times a week.

CRFK cells (ATCC CCL-94) (Crandell et al., 1973 (link)) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (10,000 units mL^–1), and streptomycin (10,000‍ ‍μg mL^–1) (Nacalai Tesque) at 37°C in a humidified atmosphere of 5% CO₂ in the air. Cells were purchased from ATCC and routinely monitored for mycoplasma contamination using the Plasmo Test™ (InvivoGen). The GFP-based FFV-infection indicator cell line, FFG, previously reported (Phung et al., 2003 (link)), was grown under the same conditions as CRFK cells.

NSC-34 cells (provided by Dr. Neil Cashman, University of Toronto, ON, Canada) were cultured to a maximum of 15 passages in a medium consisting of Dulbecco’s Modified Eagle Medium (DMEM; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies Corporation, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Life Technologies Corporation, Carlsbad, CA, USA). Cultures were incubated at 37 °C and 5% CO₂ in a humidified atmosphere. For experimentation, NSC-34 cells were seeded onto Corning BioCoat™ Poly-L-Lysine TC-Treated Culture Dishes (Corning Inc., Corning, NY, USA) at a density of 12,500 cells/cm² and incubated for 24 h according to the method described previously [16 (link)].

All cells were cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide. U87MG GBM cells (HTB14; ATCC) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Waltham, MA, USA) under standard conditions. The patient-derived glioma stem-like cell (GSC) line GSC1 that had been established in our laboratory and extensively studied in brain xenografts [18 (link)] was cultured in a serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as described previously [19 (link)].