Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

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

doi:10.3791/64603

Обогащение культур нейронов ганглиев дорсальных корешков взрослых мышей методом иммунопаннирования

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Neuroscience

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JoVE Journal Neuroscience

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Обогащение культур нейронов ганглиев дорсальных корешков взрослых мышей методом иммунопаннирования

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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.

В данной статье описывается протокол иммунопанирования для ганглиев дорсальных корешков взрослых мышей. Прикрепляя антитела к культуральным пластинам, мы можем отрицательно отбирать и удалять ненейрональные клетки. Мы показываем, что культуры обогащены для нейронов с использованием этого протокола, что позволяет углубленно изучить нейронные реакции на манипуляции.

Этот протокол позволяет увеличить количество нейронов в культурах ганглиев дорсальных корешков взрослых мышей. Это может помочь определить вклад нейронных клеток в данный ответ. Мы добавили этап иммунопанирования к базовому протоколу культивирования DRG.

Основным преимуществом является отбор против ненейрональных клеток. Если вы новичок в первичных культурах DRG, лучше всего практиковать вскрытие, чтобы получить как можно больше DRG и обрезать как можно больше нервов. Для начала аспирируйте поли-D-лизин, используемый для покрытия культуральной пластины, и трижды промойте пластину водой для культивирования тканей.

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