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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Abstract

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

Introduction

Dorsal root ganglia (DRGs) house the cell bodies of the sensory neurons which innervate peripheral tissues. Studying these neurons allows us to understand the mechanistic underpinnings of pain and sensory conditions. However, DRGs are not comprised of neurons alone, but also contain fibroblasts, Schwann cells, macrophages, and other immune cells1. Despite the presence of these various cell types, whole DRG cultures are often referred to in the literature as neuronal2,3. These cultures are still useful for neuronal investigation by imaging or flow cytometry, which would allow for neurona....

Protocol

All animal experiments were performed with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274).

1. Reagent preparation

  1. For day 1, prepare the following reagents: cell culture grade water; poly-D-lysine-prepare a stock by reconstituting the whole bottle in 50 mL of culture grade water to a stock concentration of 100 µg/mL, store at 4 °C, and dilute the stock 1:10 in culture grade water to a final concent.......

Representative Results

The fixed cells stained with DAPI and β3-tubulin were then imaged with a confocal high content screening system. Images were analyzed using suitable commercial software to determine the percentage of DAPI-positive cells that co-labelled with β3-tubulin (Figure 3). Whole DRG cultures were determined to have 42.36% ± 6.4% β3-tubulin staining, and immunopanned DRG cultures were determined to have 71.44% ±7.43% β3-tubulin staining. This reveals a significant increas.......

Discussion

This immunopanning protocol increases the proportion of neuronal cells in DRG primary cultures. For the best results, dissections should be done in a timely manner, and DRGs should be trimmed of excess nerves. The dissociation step should be carefully monitored, and should not exceed 1 h, to prevent the cells from unnecessary stress. With regard to the immunopanning specifically, each plate should be gently swirled at the halfway point to allow the cells to have access to the coated antibodies. The plates should also be .......

Acknowledgements

This work was supported by a Project Grant from the Canadian Institutes of Health Research (FRN-162434) and a Discovery Grant from the MS Society of Canada (EGID-3761). The authors would like to thank Dr. Sun and the Cross Cancer Institute for training and use of the ImageXpress system.

....

Materials

NameCompanyCatalog NumberComments
0.2 um Syringe filtersFisher723-2520
100 mm petri dishesThermoFisherFB0875712
24 well black glass-bottom platesCellvisP24-1.5H-N
70 um cell strainerCedarlane15-1070-1(BI)
B27+ supplementGibcoA3582801
Bovine Serum AlbuminSigma AldrichA7906for 15% BSA cushion, and ICC (heat shock fraction ≥98%)
Bovine Serum AlbuminSigma AldrichA4161for 4% BSA for immunopanning, and SATO for media (essentially globulin free, suitable for cell culture, ≥99%)
CD45 antibodyBD Pharmigen550539
DAPIInvitrogenD1306
DMEMGibco11960069
DNAseWorthingtonLS002007for stemxyme solution and panning buffer
D-PBSSigma AldrichD8662
EBSSSigma AldrichE6267for DNAse solution
Filter paper P8 gradeThermoFisher09-795Kfor 8% PFA
GlutamaxThermoFisher35050061
goat-anti-mouse IgGJackson Immunoresearch115-005-020for O4 dish 
goat-anti-rabbit 488InvitrogenA11008
goat-anti-rabbit IgGJackson Immunoresearch115-005-003for PDGFRB dish
goat-anti-rat IgGJackson Immunoresearch115-005-044for CD45 dish
HBSS -/-Sigma Aldrich14175145
InsulinSigma AldrichI2643
lamininInvitrogen23017-015
N-acetyl cysteineSigma AldrichA8199
NeurobasalGibco21103049
Normal Donkey SerumSigma-AldrichD9663
O4 antibodyn/an/aHybridoma
Ovomucoid trypsin inhibitorCedarlaneLS003086for low ovo
paraformaldehyde prillsSigma Aldrich441244for 8% PFA
PDGFRB antibodyAbcamAB32570
penicillin/ streptomycinGibco15140-122
Poly-D-LysineSigma AldrichP6407
ProgesteroneSigma AldrichP8783for SATO
Putrescine dihydrochrloride Sigma AldrichP5780for SATO
Sodium phosphate dibasicFisherS374-1for 0.2 M PB
Sodium Phosphate monobasic dihydrateSigma Aldrich04269for 0.2 M PB
Sodium PyruvateThermoFisher11360070
Sodium SeleniteSigma AldrichS5261for SATO
Stemxyme ICedarlaneLS004107for tissue dissociation; combination collagenase
TransferrinSigma AldrichT1147for SATO
Tris-HClMillipore SigmaT5941
trypan blueGibco15250-061
β3-TubulinSigma-AldrichT2200

References

  1. Haberberger, R. V., Barry, C., Dominguez, N., Matusica, D. Human dorsal root ganglia. Frontiers in Cellular Neuroscience. 13, 271 (2019).
  2. Perner, C., Sokol, C. L. Protocol for dissection and culture of murine dorsal root ga....

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