Neurobasal a medium

Eyes from NHPs were transferred into CO₂-independent medium (#18045-054, Thermo Fisher Scientific) after surgical removal. Upon arrival (4–5 h after surgery), the eyes were sectioned to expose the posterior eye cup, which was subsequently cut into quadrants. The retina was then gently peeled off the underlying retinal pigmented epithelium and further sectioned to separate four peripheral quadrants, which were later transferred to hydrophilic PTFE cell culture inserts with 30-mm diameter and 0.4-μm pore size (#PICMORG50, Millipore) with the ONL of the retina placed facing the membrane. Retinal explants were cultured in Neurobasal-A medium (#10888022, Thermo Fisher Scientific) supplemented with 2 mM l-glutamine (#25030024, Thermo Fisher Scientific), B27 supplement (#17504044, Thermo Fisher Scientific), and antibiotic-antimycotic (#15240062, Thermo Fisher Scientific). Retinal explants were kept overnight at 37°C, 5% CO₂ and processed for cell sorting the next day.

NPCs were differentiated into cortical neuronal cells for 16 weeks in Neurobasal A medium containing 2% B27 supplement, 1% N2 supplement, 1X GlutaMAX (all from Thermo Fisher Scientific), 200 nM l‐ascorbic acid, 20 ng/ml brain‐derived neurotrophic factor, 20 ng/ml glial cell‐derived neurotrophic factor, and 20 ng/ml neurotrophin‐3 (all from PeproTech) on PLO/laminin‐coated dishes. All media were supplemented with a 1% antibiotic–antimycotic solution (Thermo Fisher Scientific). In addition, 1 × 10⁴ cells/well were seeded on a 96‐well plate (Greiner), and the half volume of media was changed in subsequent days.

Retinas were dissected and one half cultured for 6 h in Neurobasal-A medium supplemented with 25uM glutamic acid, 2 mM glutamine, and B-27 (all ThermoFisher) and treated with DMSO as control and the other half retina cultured in identical media with 5 μm thapsigargin (ThermoFisher). Retinas were washed in PBS and RNA isolated with Trizol per manufacturer instructions. Complementary DNA (cDNA) was made using Bio-Rad cDNA kit by manufacturer instruction and 500 ng of total RNA. Reverse transcription-qPCR was performed using Bio-Rad iQ Sybr Green Supermix by manufacturer instruction with 10 ng of cDNA, and 300 nM of each of the following primers and normalized to 18 s.: XBP1sF 5’-CCA TCA CAT TGC CTA GAG GAT A-3′ and XBP1R 5’-AGC TGA GTG TCA AAC GAC AAT A-3′; ATF4F 5’-CCC CCT TCG ACC AGT CGG GT-3′ and ATF4R 5’-CCG CCT TGT CGC TGG AGA ACC-3′; ATF6F 5’-CAG ACT CGT GTT CTT CAA C-3′ and ATF6R 5’-GGC TTC TCT TCC TTC AGT-3′; p58ipkF 5’-TCC TGG TGG ACC TGC AG TACG-3′ and p58ipkR 5’-CTG CGA GTA ATT TCT TCC CC-3’ CHOPF 5’-GTC CCT AGC TTG GCT GAC AGA-3′ and CHOPR 5′-TGG AGA GCG AGG GCT TTG-3′; 18 sF 5′-GTA ACC CGT TGA ACC CCA TT-3′ and 18 sR 5’-CCA TCC AAT CGG TAG TAG CG-3′.

HSV-1 strain 17+ was originally isolated and transferred from John Hay (SUNY, Buffalo, NY, USA) to the Krause lab (FDA, Bethesda, MD, USA). HSV-2 strain 333 was originally isolated and transferred from Gary Hayward (Johns Hopkins, Baltimore, MD, USA) to the Krause lab (FDA, Bethesda, MD, USA). Virus was propagated in Vero 76 cells (CRL-1586, ATCC) in the Krause lab and Passage 1 aliquots were stored at −80 °C; most new virus stocks propagated in the Krause lab were propagated from the first passage aliquots, resulting in Passage 2. A Passage 2 aliquot was transferred to the Margolis lab (UCSF, San Francisco, CA, USA); new virus stocks were propagated and stored at −80 °C (Passage 3). A Passage 3 aliquot was transferred to the Bertke lab (Virginia Tech, Blacksburg, VA, USA); new virus stocks were propagated and stored at −80 °C (Passage 4). All virus stocks used in the Bertke lab were propagated from the Passage 4 stocks in Vero 76 cells (Passage 5). Stocks were titrated on Vero 76 cells in quadruplicate to determine concentration. Stock viruses were diluted in Neurobasal A medium (Thermo Fisher, Waltham, MA, USA) for inoculation of primary adult murine neuronal cultures.

Wild-type neuron cultures were generated from C57BL6 (Jackson Laboratories) embryos of E16.5 gestation. However, for primary mRFP-GFP-LC3 cortical neurons, transgenic mice were crossed with C57BL6 mice. At E16.5 gestation, females were sacrificed and embryos were harvested. Cortices from all embryos (regardless of genetic status) were combined to create mixed cultures^{8 (link)}.
Briefly, brains were harvested and placed in Hank’s Balanced Salt Solution (HBSS), the meninges were removed and the cerebral cortices were dissected. After incubation in HBSS with 0.25% trypsin (Gibco) for 20 min at 37 °C, dissociated neurons were resuspended in HBSS and seeded on poly-D-lysine-coated 6-multiwell or 12-multiwell plates, or MatTek glass bottom culture dishes (P35G-1.0-14-C). Cells were cultured in maintenance media (Neurobasal-A medium (#12349015, Thermo Fisher Scientific) supplemented with 2 mM GlutaMAX, 200 mM B27 supplement and 1% Penicillin–Streptomycin) at 37 °C in a humidified incubator with 5% CO₂. One half of the culture medium was changed every 2 days until drug treatment or lentivral infection. After 5 days of in vitro culture, differentiated neurons were either infected with lentiviral particles or treated with drugs as required by the experiment^{25 (link),26} .