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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.
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.
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....
All animal experiments were performed with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274).
1. Reagent preparation
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
0.2 um Syringe filters | Fisher | 723-2520 | |
100 mm petri dishes | ThermoFisher | FB0875712 | |
24 well black glass-bottom plates | Cellvis | P24-1.5H-N | |
70 um cell strainer | Cedarlane | 15-1070-1(BI) | |
B27+ supplement | Gibco | A3582801 | |
Bovine Serum Albumin | Sigma Aldrich | A7906 | for 15% BSA cushion, and ICC (heat shock fraction ≥98%) |
Bovine Serum Albumin | Sigma Aldrich | A4161 | for 4% BSA for immunopanning, and SATO for media (essentially globulin free, suitable for cell culture, ≥99%) |
CD45 antibody | BD Pharmigen | 550539 | |
DAPI | Invitrogen | D1306 | |
DMEM | Gibco | 11960069 | |
DNAse | Worthington | LS002007 | for stemxyme solution and panning buffer |
D-PBS | Sigma Aldrich | D8662 | |
EBSS | Sigma Aldrich | E6267 | for DNAse solution |
Filter paper P8 grade | ThermoFisher | 09-795K | for 8% PFA |
Glutamax | ThermoFisher | 35050061 | |
goat-anti-mouse IgG | Jackson Immunoresearch | 115-005-020 | for O4 dish |
goat-anti-rabbit 488 | Invitrogen | A11008 | |
goat-anti-rabbit IgG | Jackson Immunoresearch | 115-005-003 | for PDGFRB dish |
goat-anti-rat IgG | Jackson Immunoresearch | 115-005-044 | for CD45 dish |
HBSS -/- | Sigma Aldrich | 14175145 | |
Insulin | Sigma Aldrich | I2643 | |
laminin | Invitrogen | 23017-015 | |
N-acetyl cysteine | Sigma Aldrich | A8199 | |
Neurobasal | Gibco | 21103049 | |
Normal Donkey Serum | Sigma-Aldrich | D9663 | |
O4 antibody | n/a | n/a | Hybridoma |
Ovomucoid trypsin inhibitor | Cedarlane | LS003086 | for low ovo |
paraformaldehyde prills | Sigma Aldrich | 441244 | for 8% PFA |
PDGFRB antibody | Abcam | AB32570 | |
penicillin/ streptomycin | Gibco | 15140-122 | |
Poly-D-Lysine | Sigma Aldrich | P6407 | |
Progesterone | Sigma Aldrich | P8783 | for SATO |
Putrescine dihydrochrloride | Sigma Aldrich | P5780 | for SATO |
Sodium phosphate dibasic | Fisher | S374-1 | for 0.2 M PB |
Sodium Phosphate monobasic dihydrate | Sigma Aldrich | 04269 | for 0.2 M PB |
Sodium Pyruvate | ThermoFisher | 11360070 | |
Sodium Selenite | Sigma Aldrich | S5261 | for SATO |
Stemxyme I | Cedarlane | LS004107 | for tissue dissociation; combination collagenase |
Transferrin | Sigma Aldrich | T1147 | for SATO |
Tris-HCl | Millipore Sigma | T5941 | |
trypan blue | Gibco | 15250-061 | |
β3-Tubulin | Sigma-Aldrich | T2200 |
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