Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

Madison Trujillo; Taylor McElroy; Taurean Brown; Pilar Simmons; Fabio Ntagwabira; Anti&#241;o R. Allen

doi:10.3791/63007

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

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07:14 min

October 21, 2021

DOI: 10.3791/63007-v

Madison Trujillo^1,2,3, Taylor McElroy^1,2,3, Taurean Brown^1,2,3, Pilar Simmons^1,2, Fabio Ntagwabira^1,2,3, Antiño R. Allen^1,2,3

¹Division of Radiation Health,University of Arkansas for Medical Sciences, ²Department of Pharmaceutical Sciences,University of Arkansas for Medical Sciences, ³Neurobiology & Developmental Sciences,University of Arkansas for Medical Sciences

Overview

This protocol outlines a method for dissociating small amounts of neural tissue, allowing researchers to obtain highly viable single-cell suspensions for downstream analysis. The process is particularly useful for structure-specific studies regarding treatment efficacy and cellular functions.

Key Study Components

Area of Science

Neuroscience
Cell Biology

Background

Standard commercial kits are designed for larger sample sizes, making this protocol advantageous for low-yield samples.
Proper planning and familiarization with the protocol are emphasized to ensure successful outcomes.

Purpose of Study

To develop a reliable dissociation method for neural tissue with minimal starting material.
To facilitate detailed analysis of cellular functions and treatment mechanisms in specific brain structures.

Methods Used

The protocol employs enzymatic digestion followed by mechanical dissociation.
Utilizes isolated hippocampi from C57BL/6J mice for processing.
Involves steps like perfusion, centrifugation, and debris removal to obtain a clean cell suspension.
Centrifugation and resuspension are critical for obtaining viable cell populations.

Main Results

The method results in a higher yield of viable cells compared to manual and less optimized procedures.
It enables the calculation of cell population frequencies indicative of the neuronal compositions post-dissociation.
Findings highlight that both fresh and fixed tissues produced substantially better results than previously used methods.

Conclusions

This study validates a protocol that expands the ability to conduct cellular analyses from limited neural tissue.
Implications may extend to understanding cellular behaviors in various neurological studies and treatments.

Frequently Asked Questions

What are the advantages of this dissociation protocol?

This protocol allows for successful dissociation of small amounts of neural tissue, generating high yields of viable single-cell suspensions for analysis, surpassing limitations of commercial kits.

How is the neural dissociation performed?

The dissociation involves enzymatic digestion, followed by a mechanical dissociation process, ensuring the generation of a single-cell suspension from targeted tissue areas.

What types of data can be obtained from the dissociated cells?

The method enables quantification of cell populations, structural analysis, and assessments of cellular viability and function under various experimental conditions.

Can this method be adapted for other tissue types?

While primarily designed for neural tissue, the principles of enzymatic and mechanical dissociation may be adapted to other soft tissues in biological research.

What are the key considerations for successful tissue dissociation?

Maintaining precise techniques during perfusion and fluid transfers, along with practice prior to execution, are crucial for achieving optimal dissociation outcomes.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

Successfully dissociating small quantities of neural tissue can equip labs to gain structure-specific insight into treatment efficacy, cellular function, as well as disease and treatment mechanisms of action. This neural dissociation protocol consistently yields a highly viable and actual single-cell suspension. In addition, we can process samples that are only a fraction of those that the commercial kits are intended for.

Proper planning and preparation are key to a successful outcome for this technique. Running several practice rounds to familiarize yourself with the protocol would be beneficial. After anesthetizing a six-month-old female C57BL/6J mouse, pinch the lower abdomen and lift the skin using forceps.

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