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

Disociación mecánica y enzimática combinada del tejido del hipocampo cerebral de ratón

JoVE Journal
Neuroscience

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

Disociación mecánica y enzimática combinada del tejido del hipocampo cerebral de ratón

Full Text

4,813 Views

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

Please note that some of the translations on this page are AI generated. Click here for the English version.

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.

Este protocolo de disociación de células neuronales está destinado a muestras con una baja cantidad de material de partida y produce una suspensión unicelular altamente viable para el análisis posterior, con pasos opcionales de fijación y tinción.

Disociar con éxito pequeñas cantidades de tejido neural puede equipar a los laboratorios para obtener información específica de la estructura sobre la eficacia del tratamiento, la función celular, así como la enfermedad y los mecanismos de acción del tratamiento. Este protocolo de disociación neuronal produce consistentemente una suspensión unicelular altamente viable y real. Además, podemos procesar muestras que son solo una fracción de las que están destinados los kits comerciales.

La planificación y preparación adecuadas son clave para un resultado exitoso de esta técnica. Ejecutar varias rondas de práctica para familiarizarse con el protocolo sería beneficioso. Después de anestesiar a un ratón C57BL/6J hembra de seis meses de edad, pellizque la parte inferior del abdomen y levante la piel con fórceps.

View the full transcript and gain access to thousands of scientific videos