Biolite

MSC cells were seeded at a concentration of 1 × 10⁶ cells on fibers pre-plated on a p35 plate (Biolite, Thermo Fisher Scientific). For the maintenance of these cells, DMEM supplemented with 10% FBS, 1× P/S, and 2 mM L-glutamine was used. After 2, 4, 6, or 10 h of incubation at 37 °C and in a humidified atmosphere of 5% CO₂, micrographs of the cells were taken with a bright-field microscope (Leica DMI 3000B). To quantify the total number of cells per sample containing fibers, five representative images (20 × objective magnification) were taken of each sample, and cells were counted using ImageJ.

NKCC1-expressing COS7 cells (ATCC, Manassas, VA) were grown and maintained in 6-well plates (BioLite, Thermo Scientific), in DMEM supplemented with 10% FBS and antibiotics. Cells were cultured in 5% CO₂-95% air at 37°C. The medium was changed every 2–3 days until confluence. Plasmid transfection into COS7 cells was performed using Lipofectamine2000 following the manufacturer's instructions. Full-length human NKCC2A (hNKCC2A) cDNA cloned from human dorsal root ganglion cells (GenBank accession number EF559316) was used to create expression plasmids. The cloning plasmids harboring hNKCC2A cDNA wild type (hNKCC2A^WT) or mutants i.e., non-glycosylatable (hNKCC2A^N445/455Q) or lacking the last 181 residues (hNKCC2A^ΔC), were developed in the laboratory of one of us (FJA-L). These cDNAs were introduced into pcDNA3.1 mammalian expression vectors and FLAG-tagged. Lentiviral vectors expressing mCherry-tagged hNKCC2A^WT were from GeneCopoeia (Rockville, MD). To silence endogenous NKCC1 or NKCC2 expression, COS7 cells were stably transfected with human lentiviral constructs encoding green fluorescent protein (GFP) and shRNAs against the 4th exon of hNKCC1, as previously described (Alshahrani et al., 2015 (link); Singh et al., 2015 (link)) or against the 20th exon of hNKCC2. As control, we used constructs lacking shRNA sequences.

Prior to the guidance study, SH-SY5Y differentiation was induced by using retinoic acid (RA) and brain-derived growth factor (BDNF). For this purpose, the cells were seeded at a density of 3 × 10⁴ cells on p24 well plates treated for cell culture (BioLite, Thermo Scientific) for control, and on coverslips with the fibers with DMEM/F12 medium (Gibco) supplemented with 10% FBS, 1× P/S and 2 mM L-glutamine (d0) (growth medium, GM). After 24 h (d1), this medium was changed to DMEM/F12 medium supplemented with 1% FBS, 1× P/S, 2 mM L-glutamine, and 10 μM all-trans-retinoic acid (RA, Sigma-Aldrich) (differentiation medium 1, DM1). From this moment on, the culture was kept in the dark since the trans-isomer of RA is photosensitive. Five days later (d6), the culture medium was replaced by Neurobasal A medium (Gibco) supplemented with 1× B27 (Gibco), 1× P/S, 2 mM L-glutamine, and 50 ng/mL BDNF (Sigma-Aldrich) (differentiation medium 2, DM2). Subsequent media changes were partially performed to avoid the possible rupture of the growing axons every 3 days. Adhesion and axonal growth were studied over time until day 18, because, once the cells have differentiated into mature neurons, they can only be maintained for up to 2 weeks after terminal differentiation [22 (link)].

Chlorocebus aethiops (green monkey) kidney fibroblast COS7 cells (ATCC, Manassas, VA) were grown and maintained in 6-well plates (BioLite, Thermo Scientific) in high-glucose (25 mM) Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Cells were grown in 5% CO₂ at 37°C and media were changed every 2-3 days until ~90% confluence. To silence endogenous NKCC1 expression, early passaged COS7 cells were transfected with human lentiviral constructs encoding green fluorescent protein (GFP) and short-hairpin RNAs (shRNAs) against the fourth exon of human NKCC1 transcripts (V2LHS_93958, OpenBiosystems, Huntsville, AL). As control, we used constructs lacking shRNA sequences. Transfection was performed by using Lipofectamine 2000 and following the manufacturer's instructions. Two days after transfection, cells were washed and observed under inverted fluorescence microscope to identify GFP-expressing cells and to estimate transfection efficiency. Then, fully supplemented media containing puromycin (2.5 μg/mL) were added to initiate the selection process. Western blotting with T4 antibodies was performed to screen stably silenced GFP-expressing COS7 cells.

Plugs (7 mm diameter) bored from the margins of the isolate growing on half strength PDA medium were inoculated onto SEM in 0.2 μm-vented sterile cell culture flasks (Thermo Fisher Scientific BioLite, 130189, Loughborough, United Kingdom) that were incubated at −2, 2, 4, 9, 15, 20, and 25°C for up to 5 weeks. Four flasks were prepared for incubation at each temperature. Those at 4 and 15–25°C were incubated in refrigerators or cabinets, the temperatures of which were monitored hourly using TinyTag Plus 2 TGP-4017 loggers (Gemini Data Loggers Ltd., Chichester, United Kingdom). Those at −2, 2, and 9°C were submerged vertically into coolant in recirculating water baths (Newsham and Garstecki, 2007 (link)), with all of the flask except the neck being submerged in the coolant. Temperatures were monitored regularly using a thermometer set into SEM in a dummy flask. Measurements of the diameter of each colony recorded at 7 d intervals were subsequently used to calculate radial extension rates (mm d^–1).