Long-Term Culture of Individual <em>Caenorhabditis elegans</em> on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Luis Espejo; Bradford Hull; Leah M. Chang; Destiny DeNicola; Samuel Freitas; Vanessa Silbar; Anne Haskins; Emily A. Turner; George  L. Sutphin

doi:10.3791/64682

Cultura de Longo Prazo de Caenorhabditis elegans Individuais em Meio Sólido para Monitor...

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Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Cultura de Longo Prazo de Caenorhabditis elegans Individuais em Meio Sólido para Monitoramento Longitudinal de Fluorescência e Intervenções Aversivas

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07:25 min

December 2, 2022

DOI: 10.3791/64682-v

Luis Espejo*¹, Bradford Hull*¹, Leah M. Chang¹, Destiny DeNicola¹, Samuel Freitas¹, Vanessa Silbar¹, Anne Haskins¹, Emily A. Turner¹, George L. Sutphin¹

¹Department of Molecular & Cellular Biology,University of Arizona, Tucson

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Overview

This study presents a novel solid culture protocol for isolated nematodes, facilitating lifelong physiological tracking and fluorescence quantification. The system incorporates a palmitic acid barrier, enabling the use of aversive interventions such as pathogenic bacteria and chemical stressors.

Key Study Components

Research Area

Aging Research
Physiological Tracking
Fluorescence Imaging

Background

Current limitations in tracking individual nematode health spans.
Need for integrated systems compatible with various interventions.
Importance of understanding gene dynamics in the context of aging.

Methods Used

Solid culture of nematodes on palmitic acid barrier.
Model organism: C. elegans.
Fluorescent imaging and tracking of physiological parameters.

Main Results

Demonstrated usability of the new culture system in aging research.
Copper sulfate and dithiothreitol exposure impacted lifespan and health span differently.
Dynamic expression of KYNU-1 correlated with lifespan, highlighting intricate gene regulation.

Conclusions

The study provides a reliable protocol for tracking nematode health and longevity.
Significant implications for future research on stressors and genetic expression related to aging.

Frequently Asked Questions

What is the significance of the palmitic acid barrier?

The barrier prevents nematodes from escaping the wells, allowing controlled experiments with various stressors.

How does this method compare to traditional NGM assays?

The protocol offers comparable results while enabling automated tracking and imaging of individual nematodes.

What are the observed effects of copper sulfate and dithiothreitol?

Copper sulfate significantly reduced both lifespan and healthspan, while dithiothreitol affected lifespan without reducing health.

How does KYNU-1 expression relate to nematode longevity?

Fluorescent imaging of KYNU-1 indicates a correlation of its expression dynamics with the worms’ lifespan.

What challenges does this protocol address?

It minimizes contamination risks and enhances the reliability of physiological tracking over prolonged periods.

What kind of interventions can be tested using this system?

The system is compatible with pathogenic bacteria and chemical stressors, making it versatile for various experiments.

Why is longitudinal tracking important in this research?

Longitudinal tracking allows for better understanding of aging processes and the impact of environmental stressors on healthspan.

Aqui, apresentamos um protocolo para cultura de isolados de nematoides individuais em meio sólido para rastreamento de parâmetros fisiológicos ao longo da vida e quantificação de fluorescência. Este sistema de cultura inclui uma barreira de ácido palmítico ao redor de poços de verme único para evitar a fuga de animais, permitindo o uso de intervenções aversivas, incluindo bactérias patogênicas e estressores químicos.

Embora existam outros métodos de cultura sólida, este é o primeiro sistema de cultura sólida que permite o rastreamento longitudinal automatizado da vida e saúde animal individual e imagens longitudinais de biomarcadores fluorescentes em um sistema. Este protocolo fornece resultados comparáveis aos experimentos padrão de C.elegans em NGM sólido é compatível com intervenções aversivas e imagens diretas de fluorescência ao vivo. Este sistema será inestimável para a pesquisa do envelhecimento, especialmente no que diz respeito à exposição a longo prazo a estressores ambientais e à observação da dinâmica de genes e proteínas por longos períodos.

Evitar a contaminação e obter secagem consistente nos poços são ambos um desafio. Destacamos pontos-chave no protocolo escrito para minimizar essas questões. Para começar, triture 500 miligramas de ácido palmítico sólido em uma argamassa e pilão.

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