In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic “toolset” that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.
The protocol presented allows the measurement of C.elegans lifespan in 96 well microtiter plates. As shown in the representative results section it reliably replicates previous findings and provides quantitative information. Using this assay we have successfully screened over 89,000 molecules for their effect on C.elegans lifespan.
For the purpose of drug-screening, measuring lifespan in a 96 well microtiter plate format has several advantages over the classical solid media assay. It reduces the labor required for media preparation, the amount of incubation space, and the amount of drug required. The 96-well format and the microscopy setup allow automation of the entire assay for high throughput screenings.
During the development of the assay multiple values for each variable and combinations thereof were tested for their effects on C.elegans lifespan. These tests included different OP50 concentration ranging from 3 to 10 mg/ mL (3, 4, 6, 8, 10 mg/ mL), different number of worms per well ranging from 7 to 45 (7, 10, 15, 22, 45 worms/well), different culture volumes ranging from 40 to 150 μL per well (40, 60, 80, 100, 120, 150 μL), and changes in buffer composition. If fed with a Carbenicillin-resistant OP50, neither Carbenicillin nor Amphotericin B in the concentrations indicated were found to affect C.elegans lifespan. Continuous gentle shaking of the plates, as is often recommended in C.elegans liquid culture, was found dispensable for the small volumes used in this assay. Gentle shaking however is required if microtiter plates with larger wells are to be used. The values indicated in this protocol have been carefully selected based on statistical comparison of the different conditions tested.
The most surprising feature of this assay is probably the fact that C.elegans can be kept in 96 well plates that are sealed with tape. Side-by-side comparisons did not reveal any difference in lifespan of animals cultured in unsealed plates, in plates sealed with a plastic sealer, or in plates sealed with sealers that allow air exchange. In all three conditions, the animals developed very homogenously from L1 to gravid adults within 65 hours and showed very comparable lifespans. Animals grown in parallel on NGM developed slightly faster, but this difference was independent of the absence or presence of a sealer.
The presented protocol is based on live bacteria, but can be adapted for dead bacteria. However, in a liquid assay a single surviving bacteria can quickly multiply and re-populate the culture. In our hands, the only reliable way to kill bacteria to the extent that they can be used for liquid culture is by prolonged treatment of the bacteria with gamma irradiation.
One important point to consider in the planning of a lifespan experiment is the power of detection. Dependent on the size of the effect, the effect of the drug of interest must be tested in multiple wells. In a typical assay 4 drug-treated and 4 control wells, corresponding roughly to 40-50 animals each, should suffice to detect a 30% increase in lifespan in more than 95% of the experiments. Increases of 14 % are only detected in 60% of the cases and therefore require more replicate wells.
The wealth of lifespan data generated by this assay may be used to develop experiment or strain specific parametrical Gompertz models1. These Gompertz models are useful to determine the power of detection and to estimate the number of false positives and negatives for large-scale screens. We verified the predictions of these models in blind experiments and used them to estimate the numbers of false negatives in large screens (unpublished results).
In summary, we anticipate that the presented assay will be of great use to identify small molecules that extend lifespan of C.elegans and to study the underlying mechanisms.
The authors have nothing to disclose.
This Protocol was originally developed at Fred Hutchinson Cancer Research Center in Seattle by Xiaolan Ye and Michael Petrascheck in the Laboratory of Linda Buck. The detailed version above has been prepared to make the entire procedure available to the wider community. We thank Carol Taylor, Sarah LeBoeuf and Andy Tomacelli for critically reading the protocol. This is manuscript #2866 from The Scripps Research Institute. The Petrascheck Lab is funded by the Novartis ADI program.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Amphotericin B | Calbiochem | cat# 171375 | Also called Fungizone | |
FUDR | Sigma-aldrich | cat# F0503 | FUDR is inactivated by heat, thaw in cold water | |
Mianserin | Sigma-aldrich | M2525-250MG | Use as positive control at 50μM final concentration | |
Cyproheptadine | Sigma-aldrich | cat#C6022-MG | Alternative positive control at 10 μM final concentration | |
Sealing Tape | Nunc | cat# 236370 | Polyester, non-sterile | |
96 well plate | Falcon | cat# 351172 | Non-treated, transparent, sterile individual packaged, polystyrene | |
Nunclon flask | Nunc | cat# 178883 | used for large volumes |