Original contributionAn automated high-throughput assay for survival of the nematode Caenorhabditis elegans
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.
Section snippets
Nematode culture
Bristol N2 (wild-type), TJ1052[age-1(hx546) II], TJ1060[spe-9(hc88) I; fer-15(b26) II], TJ1062[spe-9(hc88) I; fer-15(b26) age-1(hx542) II], and E. coli OP50 were obtained from the Caenorhabditis Genetic Center at the University of Minnesota. Worms were maintained on 10 cm nematode growth medium (NGM) agar plates carrying a lawn of E. coli OP50 or in liquid culture (S-medium [18]) supplemented with E. coli OP50. Culture plates were maintained at 20°C, unless otherwise stated. Eggs were isolated
Manual thermotolerance assay using SYTOX green
To evaluate the utility of SYTOX green dye in assessing survival, 3 d old gravid adult worms were exposed to a lethal thermal stress. Wild-type and worms carrying the age-1 mutation were grown in liquid culture in 24-well plates for 3 d at 25°C (approximately 25 worms per well in duplicate). Prior to heat shock at 35°C, SYTOX green dye was added to a final concentration of 1 μM. Worms were examined by bright-field and fluorescence microscopy at 4 h after the start of the heat shock and at
Discussion
To date, more than 40 genes have been identified in C. elegans, which, when mutated, lead to an increase in life span [20]; all those tested have shown an increased resistance to acute stress 2, 20. Moreover, stress resistance has been shown to be a good surrogate measure of life span in screens for novel aging genes 12, 13, 21. Given the success of this approach, a similar strategy could be applied to screening compounds for their effect on life span if high-throughput methods with automated
Acknowledgements
This study was supported by the Brookdale National Fellowship (to M. S. G.), the Danish Research Agency (to A. O.), the Biotechnology and Biological Sciences Research Council (BBSRC) Special Studentship Program (to J. N. S.), NIH AG21069 and an Ellison Medical Foundation Senior Scholar Award (both to G. J. L.), and the Buck Institute (to A. O. and J. N. S.). The COPAS BIOSORT system was a generous gift from the Glenn Foundation for Medical Research and the Herbert Simon Foundation. All nematode
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