Source: de Luca, A. C., et al., Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration. J. Vis. Exp. (2015).
This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat's spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Experimental Set-up
2. Harvest and Dissociation of Dorsal Root Ganglia (DRG) Neurons
The authors have nothing to disclose.
100 µm cell strainer | BD Biosciences | 352360 | 70 μm strainers (ref. 352350) can be used as alternative |
15 mL plastic tubes | Sarstedt | 62.554.002 | |
50 mL plastic tubes | Sarstedt | 62.547.004 | |
75 cm2 flasks | Corning | BC301 | |
Bovine Serum Albumin (BSA) | Sigma | A9205 | |
Collagenase type IV | Worthington Biochemical | LS004188 | Note: this collagenase is only used to dissociate DRG explants |
Foetal Bovine Serum (FBS) | Biosera | FB-1001 | |
Glass pipettes | Fisher Scientific | FB50253 | Sharp material to be disposed accordingly |
Nutrient Mix F12 HAM | Sigma | N6658 | Warm at 37 °C in a water bath unless specified |
Hank's Balanced Salt Solution (HBSS) | Sigma | H9394 | Warm at 37 °C in a water bath unless specified |
N-2 supplement (100x) | Invitrogen | 17502 | |
Nerve Growth Factor 2.5s Protein, Mouse Submaxillary Glands (NGF) | Millipore | NC011 | |
Penicillin-Streptomycin (PS) | Sigma | P0781 | |
Petri dishes | Corning | 430165 | |
Trypsin (2x bovine pancreatic) | Worthington Biochemical | LS003703 | This is used for DRG dissociation |