Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

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 neuronal^2,3. 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.......

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 .......

Materials

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