1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute
Solis, G. M., Petrascheck, M. Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates. J. Vis. Exp. (49), e2496, doi:10.3791/2496 (2011).
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.
Overview: The protocol is split into four parts. Part 1 describes how to prepare the feeding bacteria. Part 2 describes how to prepare the worm culture. Part 3 describes how to score lifespan. Part 4 shows some representative results, and Part 5 describes how to prepare the required solutions. Lifespan experiments take several weeks to complete. For each step of the protocol week, day, and time of the day is included to facilitate planning. The L4 stage (Day 0) is used as the reference point for the entire protocol.
1. Preparation of Feeding Bacteria
This section describes the preparation of the feeding bacteria. The specific E. coli strain used to feed C.elegans is called OP50. Prior to this protocol, to prevent cross contamination of the worm culture with other bacteria, the OP50 strain has been made Carbenicillin/Ampicillin resistant21. Prepare the OP50 four to five days in advance. All materials coming in contact with OP50 must be sterile.
Day -7: Thursday (week 1): Inoculate 5 mL of TB containing 100 μg/ mL Ampicillin and 0.1 μg/ mL Amphotericin B with a single OP50 colony and incubate over night at 37°C in a bacterial shaker.
Day -6: Friday (week 1).
Morning 8:30. Inoculate early to allow enough time for the culture to reach saturation
2. Preparation of a Synchronous Worm Culture
This section describes the preparation of the worm culture. Its goal is to generate an age-synchronous population of worms. All materials coming in contact with C.elegans after the bleach treatment in step 5 must be sterile. Plates are kept at 20°C unless otherwise indicated.
Day -6: Friday (week 1), 4:00 p.m.: Transfer the animals to a fresh plate
Day -3: Monday (week 2) 10:00 a.m.: Establish a synchronous population
Day -2: Tuesday (week 2), 12:00 noon: Seed the animals into plates
Day 0: Thursday (week 2) before noon: Sterilize animals by adding Fluorodeoxyuridine (FUDR)
Day 1: Friday (week 2): Add drugs to culture
Day 4: Monday (week 3): Change sealers
Day 5: Tuesday (week 3): Add new OP50 to prevent starvation
3. Scoring of Lifespan.
This section describes how survival of the synchronized worm population of Part 2 is monitored until the animals died. To observe the animals in the 96 well plates, use an inverted microscope with a 2x or 2.5x objective. Record survival data three times a week; Monday, Wednesday and Friday. Use movement to determine whether the animals are alive or dead. Strong light, especially blue light, induces the animals to move. Do not remove dead animals from the assay. Occasionally, animals that did not move and were determined dead in the preceding count might move later on.
4. Representative Results.
This section shows an example of how to keep records of the lifespan data generated by this assay and some representative results.
Figure 1A shows an example of how to record lifespan data during this assay. An excel sheet is used to keep track of the survival the populations in each well. For each well it's coordinate in the plate, strain, drug, concentration of the drug and the total number of animals alive on day 0 (X0) is recorded at the beginning of the experiment. Record date, as well as the number of living and dead animals three times a week in order to follow the survival of the various populations in each well. To graph the results, calculate the fraction of animals alive for each day and plot it as a function of time in days. P-values should be calculated using statistical packages like STATA or similar software.
The medium lifespan of C.elegans is temperature dependent. Figure 1B shows that temperature dependent changes in lifespan are accurately reproduced by the microtiter based lifespan assay22. Similarly, the microtiter plate assay reproduces changes in lifespan from mutants reported to have lifespans that differ from wild type N2 animals23-24 25(Fig 1C).
The assay also can be used to make more quantitative statements. In Fig1D mean lifespan is plotted as a function of Mirtazepine concentration7. Each data point represents the mean lifespan of 7-12 population, each living in a different well. Even though the number of animals per well is relatively low (5-15 animals) the well-to-well variation is relatively small as can be seen from the error bars.
Figure 1. The microtiter plate based lifespan assay accurately reproduces changes in lifespan reported in the literature.
(A) Sample data collected in an excel spreadsheet. During each counting session date, coordinate of the well and the number of live and dead animal was recorded. At the beginning of the experiment, the total number of animals in each well was determined. (B) Survival curve of wild type (N2) animals grown at 20°C and 25°C. (C) Survival curves of strains carrying mutations that affect lifespan. All four strains were assayed in parallel at 20°C. (D) Dose response curve of Mirtazepine treated N2 animals. Mean lifespan is plotted as a function of Mirtazepine concentration. Error bars indicate the S.E.M of 8 wells per condition.
M9 buffer, 1000 mL
Potassiumphosphate buffer pH 6.0, 1000 mL
Trace metal solution
S-basal medium, 1000 mL
Potassium citrate 1M, 1000 mL
S-complete medium, 1000 mL
TB, 1000 mL
0.6 mM Fluorodeoxyuridine (FUDR, sigma cat# F0503), 1000 mL
100 mg/ mL Carbenicillin, 10 mL
250ug/ mL Amphotericin B, 4 mL
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.
No conflicts of interest declared.
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.
|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||Nalge Nunc international||cat# 236370||Polyester, non-sterile|
|96 well plate||Falcon BD||cat# 351172||Non-treated, transparent, sterile individual packaged, polystyrene|
|Nunclon flask||Nalge Nunc international||cat# 178883||used for large volumes|
Formal Correction: Erratum: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates
Posted by JoVE Editors on 05/18/2011. Citeable Link.
A correction was made to Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates. A constituent of the S-complete medium was omitted.
The omitted constituent was: