An automated high-throughput assay for survival of the nematode Caenorhabditis elegans

Abstract

Many genetic or environmental manipulations that extend life span in the nematode Caenorhabditis elegans (C. elegans) also enhance survival following acute stresses such as oxidative damage and thermal stress. This coupling of stress response and aging mechanisms has proved a useful tool in identifying new genes that affect the aging process without the need for performing lengthy life span analyses. Therefore, it is likely that this approach may also be applied to the identification of pharmacological agents that extend life span through enhanced resistance to oxygen radicals or other stressors. To facilitate high-throughput drug screens in the nematode, we have developed a microtitre plate survival assay that uses uptake of the fluorescent dye SYTOX green as a marker of nematode death. An increase in throughput compared with the conventional survival assay was achieved by combining automated worm-handling technology with automated real-time fluorescence detection. We have validated this assay by examining survival during acute heat stress and protection against oxidative stress with the superoxide dismutase/catalase mimetic Euk-134. We propose that this novel method of survival analysis will accelerate the discovery of new pharmacological interventions in aging and oxidative stress.

Introduction

Many genetic or environmental manipulations that extend life span also enhance survival following acute stress. For example, in the nematode C. elegans, mutation of the age-1 gene, encoding a phosphatidylinositol-3-kinase, extends life span and also confers resistance to a range of stresses 1, 2 including heat [3], reactive oxygen species [4], and heavy metal toxicity [5]. Such observations are evident in other species where longevity and stress resistance are also correlated 6, 7, 8. Additionally, the overexpression of specific stress response genes, such as those encoding molecular chaperones, extends life span and enhances stress resistance 9, 10, 11. These associations have been exploited in the isolation of long-lived mutants in C. elegans by selecting for their elevated stress resistance phenotype rather than performing lengthy life span analyses 12, 13. Thus, it follows that the use of a surrogate measure of longevity, such as oxidative stress resistance, could also be applied to the identification of compounds that extend life span.

Although pharmacological intervention in the aging process has been demonstrated in C. elegans 14, 15 and Drosophila melanogaster [16], the number of compounds that have been investigated remains small. High-throughput screens for other compounds that elicit a similar effect in C. elegans have been limited by the necessity for microscopic inspection of individual worms to assess survival. Touch-provoked movement is commonly used as the first indicator of death and remains the method of choice for most laboratories. This method works well for experienced investigators, yet the scoring remains somewhat subjective and labor intensive. To develop a rapid and more objective measure of survival, we have used the fluorescent dye SYTOX green (Molecular Probes, Eugene, OR, USA), which fluoresces on DNA binding yet will only enter cells whose membranes have been compromised [17]. To facilitate an increase in throughput, we have employed COPAS BIOSORT (Union Biometrica Inc., Somerville, MA, USA) automated worm-handling technology, to dispense individual nematodes into microtitre plates containing SYTOX dye, and used a fluorometric plate reader to quantify fluorescence.

In summary, we have developed an automated technique that allows for a rapid and objective score of survival in C. elegans. This system makes screening of many thousands of compounds for their effect on nematode survival a realistic proposition.