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…
Divulgaciones
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
Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells. J. Vis. Exp. (Pending Publication), e22394, doi: (2024).