Pen strep

The hearts were isolated from 8‐ to 12 ‐week‐old Cas9⁺ mice and transferred to sterile cold Hank's balanced salt solution (HBBS; Sigma) + 2x Pen/Strep (Life Technologies). The hearts were then dissected, rinsed, and finely minced before transfer into digestion buffer prewarmed to 37°C (980 U/ml collagenase II [Life Technologies], 20 KU/ml DNase I [Stem Cell Technologies] in HBSS + 1x Pen/Strep) for 10 minutes. Digestion was then centrifuged for 5 minutes at 600g and incubated with 5 ml TryPLE (Life Technologies) for 5 minutes. Entire pellet was resuspended in 4.5 ml prewarmed CF media (15% fetal bovine serum [FBS], 1% NEAA, 1x Pen/Strep in Dulbecco's modified Eagle's medium [DMEM]+GlutaMAX [Life Technologies]) and passed through a 40 μm filter to remove any large undigested pieces. Cell suspension was plated onto four wells of a 0.1% gelatin‐coated six‐well plate. Plate was incubated overnight at 37°C and media was exchanged the following day. When confluent, cells were passaged once with 0.5% trypsin‐EDTA (Life Technologies) before being frozen down at 3 × 10⁵ cells per milliliter in 90% FBS + 10% dimethyl sulfoxide (DMSO; Sigma‐Aldrich).

On the day of culture, plating media with supplements was prepared: NeuroCult^™ Plating Media (Stem Cell Technologies, 05713), 1X SM1 neuronal supplement (Stem Cell Technologies, 05711), 500μM L-Glutamine (Gibco, 25030149), and 100unit Penicillin/100μg Streptomycin (Pen/Strep) (ThermoFisher, 15140) (referred to as plating media hereon).
Retinal tissue was dissected as described above. A hydrophilic PTFE cell culture insert (Millicell, PICM0RG50) was prepared by pipetting 150uL of plating media onto insert membrane. Retinal tissue was mounted onto the PTFE membrane with the retinal apical surface (photoreceptor side) in juxtaposition. Tissue was placed into a 6-well plate (Corning, 353004) containing 1mL plating media and incubated at 37°C with 5% CO². After 48 hours, 500uL of initial plating media was replaced with BrainPhys^™ Neuronal Medium (Stem Cell Technologies, 05790) containing 1X SM1 neuronal supplement, 1X N2 Supplement-A (Stem Cell Technologies, 07152) and Pen/Strep (referred to as BrainPhys media hereon). 500μL of media was removed and replaced with fresh BrainPhys media every 48 hours for the extent of the culture.

Human induced pluripotent stem cells (hiPSCs) (IMR90-4/WISCi004-B, WiCell) were cultured on human laminin 521 (Biolamina)-coated culture treated six-well plates and maintained in mTeSR+ medium (STEMCELL Technologies) supplemented with 1% (v/v) Pen-Strep. Cells were maintained at 37 °C with 5% CO₂, passaged weekly with ReLeSR™, and subcultured in mTeSR+ medium with 10 µM Y-27632 Rho Kinase (ROCK) inhibitor (Cayman Chemical Company). Lines were kept in culture with the removal of differentiated patches when necessary and regular testing for mycoplasma was performed. The maintenance and subsequent experiments with hiPSCs during maintenance and experiments were performed in accordance with relevant guidelines and regulations.

Dissociated hippocampal cultures were prepared from embryonic day 18 (E18) rat brains of both genders, as described in^{42 (link)} and in accordance to the approved DEC work-protocol as mentioned in the ethics statements above. Mother rats were sacrificed by gradual fill CO2/O2. Subsequently the uterus containing the pups is taken out and is stored in a sterile ice cold environment. After the pups were sedated by the cold, they were removed from the uterus and decapitated. Dissociated neurons were plated on Ø18-mm coverslips coated with poly-L-lysine (37.5 µg/ml, Sigma-Aldrich) and laminin (1.25 µg/ml, Roche Diagnostics) at a density of 100,000 neurons per well. Neurons were grown in Neurobasal medium (NB) supplemented with 1% pen/strep, 2% [v/v] B27, and 0.5 mM L-glutamine (all from Gibco) (NB-complete medium) at 37 °C in 5% CO_2. From days in vitro (DIV) 1 onward, medium was refreshed weekly by replacing half of the medium with Brainphys neuronal medium (BP) supplemented with 2% [v/v] NeuroCult SM1 neuronal supplement (STEMCELL Technologies) and 1% pen/strep (BP-complete medium).

A Jurkat T-cell line containing a mixed population of latent HIV clones (here referred to as Jurkat T cells) was obtained from Jonathan Karn (Case Western Reserve University, Cleveland, OH) and cultured in RPMI 1640 media with 5% fetal bovine serum (FBS), 100 U/ml penicillin (Pen)-streptomycin (Strep), and 2 mM l-glutamine (Thermo Fisher, Carlsbad, CA). HCT116 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in McCoy’s 5a medium modified with 10% fetal bovine serum (FBS) and 100 U/ml Pen-Strep. Primary resting CD4 T cells were isolated from PBMCs from four healthy donors (Biological Specialty Corporation, Colmar, PA). Resting CD4 T cells were isolated in 2% FBS in phosphate-buffered saline (PBS) by negative magnetic selection using an EasySep custom kit (Stemcell Technologies, Cambridge, MA) containing the following antibodies: anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD56, anti-CD25, anti-CD41, and anti-HLA-DR, plus glyphorin A. After isolation, CD4 T cells were cultured in RPMI 1640 media with 10% FBS and 100 U/ml Pen-Strep at 5 × 10⁶ cells/ml.

Cortices of mice at embryonic day 14.5 (E14.5) for transfection and TrkB phosphorylation assays and E17.5 for immunostaining studies were collected in Hanks’ buffered salt solution (Sigma-Aldrich) and trypsinized in 1 mg/ml trypsin (Worthington) for 20 min at 37°C. The reaction was then stopped using 1 mg/ml trypsin inhibitor (Sigma-Aldrich) before the addition of 1 mg/ml DNase I (Thermo Fisher Scientific) and gentle dissociation with a 5 ml serological pipette. Cells were then pelleted by centrifugation at 1400 rpm for 5 min and resuspended in DMEM supplemented with 2% FBS, 1% GlutaMAX, and 1% Penstrep. Three hours after plating into wells coated with poly-d-lysine (Sigma-Aldrich), cells were maintained in Neurobasal medium supplemented with 1% GlutaMAX supplement, 1% Penstrep, and 2% SM1 supplement (Stem Cell Technologies). Neurons were cultured for up to 12 d with 50% media changes performed three times weekly. Subsequent transfections were performed on E14.5 neurons at 5DIV using 0.5 μg of indicated DNAs and 1 μl of Lipofectamine 2000 (see above). Depolarization of E17.5 neurons at DIV11 was achieved by supplementing media with 1 mm 4-aminopyridine (4-AP; Merck) for 24 h.