Materials and reagents

All materials, reagents, and solutions should be sterile and cell culture grade. If possible, consistently use materials and reagents of the same brand and maintain a record of lot numbers in case signs of cell toxicity are observed. The list below is not intended to be fully comprehensive or limit the use of products from certain manufacturers. The product information is provided for reference only. Follow the manufacturer’s recommendations and use best practices in cell culture to avoid microbial contamination and ensure the reproducibility of the results.

Supplies and consumables

1. Disposable serological pipettes (5, 10, and 25 mL), polystyrene, plugged, sterile, and individually wrapped (VWR, catalog numbers: 76201-710, 75816-100, and 75816-090)

2. Polystyrene Pasteur (transfer) pipettes, sterile and individually wrapped (VWR, Argos Technology, catalog number: 10122-560)

3. Petri dish, bacteriological grade, 10 cm, not tissue culture treated (Corning, catalog number: 351029)

4. Centrifuge tubes, 15 and 50 mL, polypropylene, conical-bottom (Corning, catalog numbers: 430791 and 430290)

5. Cell culture dishes (35, 60, and 100 mm), polystyrene, tissue culture treated (Corning, catalog numbers: 353001, 353002, and 353003)

6. Multi-well plates (6, 12, and 24-well), tissue culture treated, flat bottom (Corning, catalog numbers: 3506 and 3524)

7. Cell culture flasks (T25 and T75), canted neck with plug seal caps (Corning, catalog numbers: 430168 and 430720U)

8. Cryogenic vials, 2 mL, internal thread (Corning, catalog number: 430489)

9. Polycarbonate freezing container with a tube holder, Nalgene “Mr. Frosty” Freezing Container (VWR, catalog number: 55710-200)

10. Cell lifter, individually wrapped, polyethylene, 2 cm length blade, 18 cm long (VWR, catalog number: T-2443-4)

11. Ice pans (VWR, catalog number: 89233) containing wet ice and dry ice

Media, supplements, and other cell culture products

1. Distilled water, cell culture grade (Fisher Scientific, Gibco, catalog number: 15-230-147)

2. Hank’s balanced salt solution (HBSS), formulated without calcium or magnesium and containing phenol red, pH 7.2 (Thermo Fisher Scientific, Gibco, catalog number: 14170-112)

3. Leibovitz’s L15 Medium (L15) (Thermo Fisher Scientific, Gibco, catalog number: 11415064)

4. Dulbecco’s Modified Eagle’s Medium (DMEM), with high glucose and phenol red, pH 7.2 (Thermo Fisher Scientific, Gibco, catalog number: 11965092). (Optional) Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Thermo Fisher Scientific, Gibco, catalog number: 11320033)

5. Gentamycin 1,000× provided as 50 mg/mL stock solution (Thermo Fisher Scientific, Gibco, catalog number: 15750-060). (Optional) Penicillin-Streptomycin, 10,000 U/mL stock solution (Thermo Fisher Scientific, Gibco, catalog number: 15140-148)

6. De-complemented fetal bovine serum (FBS) (HyClone, catalog number: SV 30014.03). Stored in aliquots at -80 °C; used in all applications requiring serum, including cryopreservation of cell stocks

7. GlutaMAX supplement (100×) consisting of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl (Thermo Fisher Scientific, Gibco, catalog number: 35050061)

8. Heregulin-β1, 177-244 amino acid peptide (Preprotech, catalog number: G-100-03); instructions on the preparation, storage, and use of heregulin-β1 stock solution (25 µM) can be found in our protocol Andersen and Monje (2018). The stock solution is best preserved when kept in aliquots at -80 °C and used only for media preparation, avoiding freezing/thawing cycles. Heregulin-β1 is the primary mitogenic factor for hSCs; it is referred to as “heregulin” in the text and figures

9. Forskolin powder (Sigma-Aldrich, catalog number: F68861); instructions on the preparation, storage, and use of forskolin stock solution (15 mM) can be found in our protocol Andersen and Monje (2018). Keep the forskolin stock solution in the -80 °C freezer for long-term preservation of the cAMP-inducing activity. Forskolin is a synergistic enhancer of heregulin-dependent hSC proliferation

10. 0.5% Trypsin/EDTA (TE) solution (10×), without phenol red (Thermo Fisher Scientific, Gibco, catalog number: 15400054). (Optional) TrypLE^TM select enzyme, 1× (Thermo Fisher Scientific, Gibco, catalog number: 12563011)

11. Laminin stock, consisting of a sterile 1 mg/mL laminin solution from Engelbreth-Holm-Swarm murine sarcoma basement membrane (Sigma-Aldrich, catalog number: L2020), stored at -80 °C in aliquots for single use. Prepare laminin-coated dishes as described in Andersen and Monje (2018). Always use freshly coated laminin dishes for seeding hSCs. Nerve fibroblasts can be plated in regular cell culture–treated dishes without coating

12. Poly-L-lysine (PLL) powder (Sigma, catalog number: P-263). Prepare a PLL stock solution and use it for coating dishes as described in Andersen and Monje (2018). Store air-dried PLL-coated dishes at 4 °C for up to one month

13. Dulbecco’s phosphate-buffered saline (DPBS), pH 7.2 (Thermo Fisher Scientific, Gibco, catalog number: 14190)

14. Dimethyl sulfoxide (DMSO) (Invitrogen, catalog number: D12345). Used as a component of the freezing medium and solvent of lyophilized fluorophores

15. (Optional) Recovery^TM cell culture freezing medium, ready to use, stored in aliquots at -80 °C (Thermo Fisher Scientific, Gibco, catalog number: 12648010)

16. Isopropyl alcohol (Fisher Chemical, catalog number: A416P-4L)

17. Tris base, powder (Thermo Fisher Scientific, Invitrogen, catalog number: 15504020)

18. Hydrochloric acid (HCl) solution, 1 N, bioreagent suitable for cell culture (Sigma, catalog number: 7647-01-0); used to balance pH in buffers and cell culture medium

Antibodies, dyes, and assorted commercially available products

1. Anti-NGFR mouse IgG monoclonal antibody, produced from the HB-8737 hybridoma cell line (American Type Culture Collection, ATCC). See Ravelo et al. (2018) for technical details on our culture protocols for this and other hybridoma cell lines

2. Anti-O4 mouse IgM monoclonal antibody, produced from the O4 hybridoma cell line (Sommer and Schachner, 1981), kindly provided by Dr. Melitta Schachner. (Optional) Use purified O4 antibodies from a commercial source (Novus Biologicals, catalog number: NL637)

3. Anti-mouse immunoglobulins, goat polyclonal antibody (anti-IgG, IgA, and IgM), affinity purified, liquid, unconjugated (ICN/Cappel, catalog number: 55486)

4. CellTracker^TM Green CMFDA (5-chloromethylfluorescein diacetate) powder (Invitrogen, Molecular Probes, catalog number: C7025)

5. (Optional) Pluronic F-127 (Molecular Probes, catalog number: P-6866)

6. Hoeschst-34580 (Molecular probes, catalog number: H21486) prepared in water at 1 mg/mL

7. Basic Nucleofector^TM kit for primary mammalian glial cells (Lonza, catalog number: VPI-1006)

8. GFP plasmids. The pmaxGFP^TM expression vector (0.5 µg/µL in 10 mM Tris pH 8.0) can be used as provided in the Basic Nucleofector^TM kit or expanded in house

9. Lentiviral particles, aliquoted and stored at -80 °C for up to six months. For a reference, we have used lentiviral expression vectors for the fluorescent reporters EGFP and mCherry (Monje et al., 2018) acquired as ultra-purified, ready-to-use, packaged lentiviruses from the Viral Vector Core Facility, The Miami Project to Cure Paralysis, Miami, FL. Follow the manufacturer’s recommendations on the safe storage and use of viral stocks, including the MOI determination for each virus lot. Multiple freeze/thaw cycles of the viral particles are not recommended

10. (Optional) Polybrene infection/transfection reagent, 10 mg/mL stock solution (Santa Cruz Biotechnology, catalog number: sc-134220)

11. (Optional) Eukaryotic antibiotics for the selection of virally-infected cells. Puromycin dihydrochloride (Santa Cruz Biotechnology, catalog number: sc-108071); Blasticidin S HCl solution (Santa Cruz Biotechnology, catalog number: sc-495389); Hygromycin B solution (Santa Cruz Biotechnology, catalog number: sc-29067)

1. Low proliferation medium (LP) (see Recipes)

2. High proliferation medium (HP) (see Recipes)

3. TE dissociation solution (see Recipes)

4. Freezing medium (see Recipes)

5. Immunoglobulins solution (see Recipes)

Equipment

1. Biosafety cabinet, BL2 level (Thermo Fisher Scientific, model: 1300 Series A2)

2. CO₂ cell incubator set up at 37 °C and 8%–9% CO₂ (Thermo Fisher Scientific, model: Forma Steri-Cycle)

3. Benchtop centrifuge (Beckman Coulter, model: Allegra X-I2R)

4. Inverted phase contrast microscope with an attached digital camera (VWR, model: V5MP)

5. Cell counter for automated counting of cells in suspension (Bio-Rad, TC20). (Optional) Hemocytometer for manual cell counting

6. 4 °C refrigerator and -80 °C laboratory freezer (Thermo Fisher Scientific, model: RLE Series)

7. Liquid nitrogen storage tank (Thermo Fisher Scientific, model: Locator JR Plus)

8. Single cuvette-based Nucleofector^TM I device and certified cuvettes (formerly Amaxa Biosystems, now Lonza)

9. Inverted fluorescence microscope equipped with a UV, FITC, and TRITC filter sets (Olympus IX71) and attached digital camera

10. (Optional) Live-cell imaging system IncuCyte ZOOM^TM (Essen BioScience, MI) for time-lapse microscopy of cultured cells plated in multi-well dishes

Software

1. (Optional) IncuCyte Zoom 2015A, Rev 1 for image capture and video assembly of time-lapse microscopy data

2. Image analysis software, ImageJ-FIJI (NIH), free-access software available at https://imagej.net/software/fiji/. Used to estimate the covered area (confluency) in selected phase contrast images using the area measuring tool

Procedure