Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells
Published: July 31, 2024
Abstract
Source: Tsui, Y, et al. Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells. J. Vis. Exp. (2017).
This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.
Protocol
Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons 1. Preparation of purified rat DRG neurons Autoclave all dissection tools (i.e., dissection scissors, forceps, two microdissection forceps, and microdissecting scissors) at 180 °C for at least 2 h prior to use. Coat 6-well tissue culture plates with poly-D-lysine (PDL, 10 µg/mL in PBS) at 4°C overnight. Remove the PDL and rinse with 1.5 m of PBS per w…
Representative Results
Figure 1: Establishing purified rat DRG networks. Purified DRG networks are established after pulsed treatment with the antimitotic agents FDU and uridine (A). Neurite networks that are devoid of S100β-expressing endogenous glia (B) are ready for coculture with SCLCs. Scale bars = 100 µm. <img…
Offenlegungen
The authors have nothing to disclose.
Materials
| αMEM | Sigmaaldrich | M4526 | |
| DMEM/F12 | Thermofisher scientific | 12400-024 | |
| Neurobasal medium | Thermofisher scientific | 21103-049 | |
| FBS | Biosera | FB-1280/500 | |
| B27 | Thermofisher scientific | 17504-001 | |
| Epidermal growth factor (EGF) | Thermofisher scientific | PHG0313 | |
| Basic fibroblast growth factor (bFGF) | Peprotech | 100-18B/100UG | |
| Nerve growth factor (NGF) | Millipore | NC011 | |
| Platelet-derived growth factor-AA (PDGF-AA) | Peprotech | 100-13A | |
| Heregulin beta-3, EGF domain (β Her) | Millipore | 01-201 | |
| Uridine | Sigmaaldrich | U3003 | |
| 5-Fluro-2' – deoxyuridine (FDU) | Sigmaaldrich | F0503 | |
| Poly-D-lysine (PDL) | Sigmaaldrich | P7886-1G | |
| Laminin | Thermofisher scientific | 23017015 | |
| GlutaMAX | Thermofisher scientific | 35050061 | |
| Penicillin / streptomycin (P/S) | Thermofisher scientific | 15140-122 | |
| TrypLE Express | Thermofisher scientific | 12604-013 | |
| 10 cm plate for adherent culture | TPP | 93100 | Used for selection of MSCs by tissue culture adherence |
| 6-well plate for adherent culture | TPP | 92006 | Used for expansion of MSCs following passaging |
| UltraLow 6-well plate for non adherent culture | Corning | 3471 | Used for neural progenitor enrichment |
| Hypoxia chamber | Billups-Rothenberg | MIC-101 | |
| HEPES buffer | Sigmaaldrich | H4034-100G |
Diesen Artikel zitieren
Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells. J. Vis. Exp. (Pending Publication), e22394, doi: (2024).