Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Aislinn  D. Maguire; Jason R. Plemel; Bradley J. Kerr

doi:10.3791/64603

Yetişkin Fare Dorsal Kök Ganglia Nöron Kültürlerinin İmmünupanjör ile Zenginleştirilmesi

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Neuroscience

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JoVE Journal Neuroscience

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Yetişkin Fare Dorsal Kök Ganglia Nöron Kültürlerinin İmmünupanjör ile Zenginleştirilmesi

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08:57 min

February 24, 2023

DOI: 10.3791/64603-v

Aislinn D. Maguire¹, Jason R. Plemel^1,2,3, Bradley J. Kerr^1,4,5

¹Neuroscience and Mental Health Institute,University of Alberta, ²Department of Medicine, Division of Neurology,University of Alberta, ³Department of Medical Microbiology and Immunology,University of Alberta, ⁴Department of Pharmacology,University of Alberta, ⁵Department of Anesthesiology and Pain Medicine,University of Alberta

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Overview

This paper details an immunopanning protocol for enriching neurons from adult mouse dorsal root ganglia (DRG) cultures. The method negatively selects against non-neuronal cells, facilitating a focus on neuronal responses to specific conditions in the cultures.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neuronal culture techniques

Background

Dorsal root ganglia (DRG) contain sensory neurons from the peripheral nervous system.
Isolating neurons from DRG is crucial for studying their physiological responses.
Conventional culture methods often lead to contamination by non-neuronal cells.
This study aims to refine these methods for improved neuronal enrichment.

Purpose of Study

To develop an effective immunopanning protocol for cultured DRGs.
To enhance neuronal yield from adult mouse DRGs.
To facilitate the investigation of neuronal behavior in vitro.

Methods Used

Cell culture techniques in vitro using adult mouse dorsal root ganglia.
The biological model used is primary sensory neurons isolated from DRGs.
An immunopanning approach was incorporated to selectively isolate neuronal cells.
Critical steps involve careful dissection and enzymatic treatment to dissociate neurons.
The method focuses on minimizing non-neuronal cell presence through antibody-coated plates.

Main Results

The immunopanning protocol resulted in a substantial increase in the proportion of neurons.
Cultured neurons showed improved viability and reduced contamination from non-neuronal cells.
Enrichment assessments revealed a notable rise in beta3-tubulin staining, indicating higher neuronal content.

Conclusions

This study demonstrates a robust method for enriching neurons from adult mouse DRG cultures.
The protocol enables enhanced examination of specific neuronal responses and properties.
It holds implications for understanding neuronal behaviors and mechanisms in various research contexts.

Frequently Asked Questions

What is the main advantage of the immunopanning protocol?

The immunopanning protocol effectively reduces non-neuronal cell contamination, leading to a more enriched neuronal culture, which is essential for focused studies on neuronal physiology.

How are the dorsal root ganglia prepared for culture?

The DRGs are isolated from euthanized mice using careful dissection techniques, followed by enzymatic dissociation to enable single-cell cultures.

What kind of data can be obtained from this experiment?

Data includes neuronal viability, enrichment ratios, and physiological responses of cultured neurons, assessed via beta3-tubulin immunostaining.

Can this method be adapted for other types of neurons?

Yes, while this protocol is optimized for DRGs, it can potentially be adapted for other sensory or peripheral neurons with adjustments to culture conditions.

What are some limitations, if any, of the immunopanning method?

The main limitation could be the potential loss of some neuronal subtypes during the selection process and the need for meticulous handling during tissue preparation.

Bu yazıda yetişkin fare dorsal kök gangliyonları için bir immünopanning protokolü açıklanmaktadır. Antikorları kültür plakalarına yapıştırarak, nöronal olmayan hücreleri negatif olarak seçebilir ve çıkarabiliriz. Kültürlerin bu protokolü kullanarak nöronlar için zenginleştirildiğini ve manipülasyona karşı nöronal tepkilerin derinlemesine incelenmesine izin verdiğini gösteriyoruz.

Bu protokol, yetişkin fare dorsal kök gangliyon kültürlerinde nöron sayısını arttırmamızı sağlar. Bu, nöronal hücrelerin belirli bir cevaba katkısını belirlemeye yardımcı olabilir. Temel DRG kültür protokolüne bir immünopanning adımı ekledik.

Başlıca avantajı, nöronal olmayan hücrelere karşı seçim yapmaktır. Birincil DRG kültürlerinde yeniyseniz, mümkün olduğunca çok DRG elde etmek için diseksiyon uygulamak ve sinirin mümkün olduğunca çoğunu kesmek en iyisidir. Başlamak için, kültür plakasını kaplamak için kullanılan poli-D-lizini aspire edin ve plakayı doku kültürü suyuyla üç kez yıkayın.

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