Materials and reagents

Our cell culture protocols use research-grade materials, reagents, and solutions that are endotoxin-free and suitable for cell culture. Still, researchers are encouraged to assess the quality of relevant materials by appropriate methods to identify potentially toxic or incompatible elements in the culture medium, coating reagents, and plasticware.

All assays can be implemented using basic cell culture equipment and labware typically available in research labs. The description of products provided below is for reference only. Researchers may find comparable items from other providers. An exception is the primary antibodies (Table 1) because they have been validated carefully in our lab using cultured hSCs and nerve tissues. If other antibodies are selected, take into consideration that the ones used for rodent SC research may fail to recognize the respective antigens from humans. For this reason, it is important to confirm the specificity and reactivity of all antibodies in-house using proper positive and negative controls. Some of the suggested monoclonal antibodies were produced in our laboratory from hybridoma cell cultures but alternative ones are available from commercial sources (see Table 1). Lastly, the list below is not fully comprehensive. Additional technical details can be found in the accompanying papers (Aparicio and Monje, 2023; Monje, 2023).

Table 1. Useful antibodies to characterize human Schwann cells (hSCs) and non-glial cells established in cell culture. These antibodies were validated using traditional adult nerve-derived hSC monocultures. Staining with NGFR, Sox10, and S100B antibodies, alone or together with fibronectin, SMA (α-smooth muscle actin), and FAP (fibroblast activation protein/seprase) antibodies, is recommended for the initial characterization of hSCs and non-glial cells. We have not found a ubiquitous non-glial cell marker. Protocol 1C is suitable for staining with all antibodies except for anti-O1 and anti-O4, which can be accomplished only in live cells (Protocol 1B). A 1:200–1:500 starting dilution is suitable for most antibodies; the optimal concentration should be determined by the end user in the target cells. (*) Indicates that the antibodies are produced from hybridoma cell lines. MBP: myelin basic protein; MAG: myelin-associated glycoprotein; MPZ: myelin protein zero; GFAP: glial fibrillary acidic protein.

Supplies and consumables

1. Polypropylene conical-bottom centrifuge tubes, 15 and 50 mL (Corning, catalog numbers: 430791 and 430290)

2. Serological pipettes, 5, 10, and 25 mL, polystyrene, sterile (VWR)

3. Pasteur pipettes, polystyrene, individually wrapped for liquid disposal (VWR, Argos Technology, catalog number: 10122-560)

4. Laminin-coated cell culture dishes for cell expansion. 100 mm × 20 mm plates, polystyrene (Corning, catalog number: 353003) coated with a laminin substrate, as described in Andersen and Monje (2018)

5. Laminin-coated multi-well plates for analytical assays. Cell culture–treated 24-well plates, flat bottom, polystyrene (Corning, catalog number: 3524) coated sequentially with PLL and laminin, as described in (Andersen and Monje, 2018). (Optional) Use commercially available 24-well plates coated with poly-L-ornitine (PO) and laminin (BD Biosciences, catalog number 354659). Do not plate hSCs on uncoated surfaces. Polystyrene plates or chamber slides are preferred. Coverslips are not suitable since the hSCs are unstable and display an abnormal morphology on any glass surface

6. Paraformaldehyde (PFA) 20% stock solution (Electron Microscopy Sciences, catalog number: 15713)

7. Methanol (Sigma, catalog number: 154903) maintained at -20 °C for cell permeabilization. (Optional) 0.1% (v/v) Triton X-100 (Sigma, catalog number: 11332481001) prepared in D-PBS and stored at 4 °C

8. Laboratory wrapping film (Parafilm, catalog number: PM-996) and aluminum foil

Media, supplements, and other cell culture products

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

2. Dulbecco’s phosphate-buffered saline (DPBS) with calcium and magnesium, pH 7.2 (Thermo Fisher Scientific, Gibco, catalog number: 14190)

3. 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)

4. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and phenol red, pH 7.2 (Thermo Fisher Scientific, Gibco, catalog number: 11965092)

5. DMEM, Nutrient Mixture F-12 (DMEM/F-12), no glutamine, with phenol red (Thermo Fisher Scientific, Gibco, catalog number: 21331020)

6. De-complemented fetal bovine serum (FBS) (HyClone, catalog number: SV 30014.03), stored in aliquots at -80 °C

7. 100× GlutaMAX supplement (Thermo Fisher Scientific, Gibco, catalog number: 35050061)

8. Gentamycin 50 mg/mL, 1,000× stock solution (Thermo Fisher Scientific, Gibco, catalog number: 15750-060)

9. HEPES buffer solution 1 M (Thermo Fisher Scientific, Gibco, catalog number: 15630-080)

10. Normal goat serum (GeneTex, catalog number: GTX73206), stored in aliquots at -80 °C

11. Forskolin (Sigma-Aldrich, catalog number: F68861); for a detailed protocol on preparation, storage, and use of forskolin stock solution, see Andersen and Monje (2018)

12. Heregulin-β1 (referred to as heregulin), HRG1-B1177-244 recombinant peptide (Preprotech, catalog number: G-100-03); for a detailed protocol on preparation, storage, and use of heregulin stock solution, see Andersen and Monje (2018)

13. CPT-cAMP stock solution (5 mM in DMEM), prepared from adenosine 3′,5′-cyclic monophosphate, 8-(4-chlorophenylthio), sodium salt (Calbiochem, catalog number: 116812); for a detailed protocol on preparation, storage, and use of CPT-cAMP stock solution, see Monje (2018)

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

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

16. Starvation or D1 medium (DMEM/F12 - 1% FBS) (see Recipes)

17. PFA-based fixation solution (see Recipes)

18. Blocking solution (see Recipes)

Antibodies, dyes, and commercially available detection kits

1. Mouse monoclonal antibodies from hybridoma cell lines. Anti-nerve growth factor receptor (NGFR), anti-O4, and anti-O1 (Sommer and Schachner, 1981) in the form of conditioned medium produced from HB-8737 cells (also known as 200-3-G6-4, obtained from the American Type Culture Collection, ATCC), O4 cells, and O1 cells (kindly provided by Dr. Melitta Schachner), respectively. Researchers can refer to our publication (Ravelo et al., 2018) for technical details on our hybridoma culture protocols. Briefly, transfer the cell content of a hybridoma stock (1 × 10⁶–2 × 10⁶ cells/cryovial) directly into a T-75 flask containing Iscove’s modified Dulbecco’s medium (with phenol red) supplemented with 10% FBS and antibiotics. Culture the cells in suspension inside a CO₂ incubator until the cultures are sufficiently dense and the medium becomes slightly acidic. Next, separate the culture supernatant from the cellular content by centrifugation to obtain conditioned medium enriched in monoclonal antibodies. The conditioned medium is often used without dilution, but the specificity and reactivity of each batch should be tested using appropriate positive control cells or tissues

2. Primary antibodies. pERK1/2/MAPK mouse monoclonal antibody (Santa Cruz, catalog number: sc7383); pAkt-Ser-473 rabbit polyclonal antibody (Santa Cruz, catalog number: sc7985); Akt rabbit polyclonal (Cell Signaling, catalog number: 9272); ERK2/MAPK rabbit polyclonal (Santa Cruz, catalog number: sc154); human nuclei (HNA), mouse monoclonal (Sigma, MAB1281, clone 235-1) or HNA mouse monoclonal (Abcam, ab191181). Other antibodies are listed in Table 1

3. Fluorescent secondary antibodies of the appropriate species and class, Alexa Fluor^TM-conjugated (Molecular Probes). Fluorochromes should be chosen and combined as optimal for visualization

4. FM4-64FX, fixable membrane stain (Invitrogen, catalog number: F34653). (Optional) FluoroMyelin^TM (red or green) myelin stain (Invitrogen, catalog number: F34652)

5. Hoeschst-34580 (Molecular probes, catalog number: H21486). (Optional) 4′,6-Diamidino-2-Phenylindole, Dilactate, (DAPI, Invitrogen, catalog number: D3571)

6. Click-iT EdU (5-ethynyl-2′-deoxyuridine) Alexa-Fluor-594 Imaging Kit (Life Technologies, catalog number: C10339)

7. Senescence-associated (SA) β-Galactosidase (SA-β-Gal) staining kit (Cell Signaling Tech, catalog number: 9860)

8. VectaShield^TM antifade liquid mounting reagent for fluorescence (Vector Laboratories, catalog number: H-1000). (Optional) Prepare a homemade mounting reagent, as suggested in Ravelo et al. (2018), to control photobleaching and preserve the stained cultures. Add a sufficient volume of mounting reagent to fully cover the fixed cells (300 μL/well in a 24-well plate) or a couple of drops if a glass coverslip is mounted on top

Equipment

1. Biological safety cabinet, BL2 level (Thermo Scientific 1300 series class II, 1300 series, type A2)

2. Cell incubator set at 37 °C and 8%–9% CO₂ (Thermo Scientific Forma, series II, water-jacketed)

3. Inverted phase contrast microscope (VWR) equipped with 10× to 40× phase contrast objectives and attached digital camera (VWR, V5MP)

4. Benchtop refrigerated centrifuge (Beckman Coulter^TM, Allegra X-12R) equipped with swing bucket rotor (SX4750) and adapters for 50 and 15 mL tubes

5. Automated cell counter for the image-based counting of cells in suspension (Bio-Rad, TC20) or a hemocytometer for manual cell counting

6. Inverted fluorescence microscope (Olympus, IX71) for brightfield, phase contrast, and fluorescence microscopy; equipped with standard UV, FITC, and TRITC filter sets and attached digital camera

Procedure