In this five part series we are exploring some common mishaps when working with C. elegans, so you can hopefully avoid them too! Especially since when testing a specific variable (compound, strain, etc.) we want to limit all other variables that may skew results or lead to inaccurate data In Part 4 we ask: why am I getting inconsistent results from a drug treatment I am trying? How do I know my drug is being absorbed or eaten by the worms? Resveratrol was supposed to extend lifespan of my worms, why were the results not significant from the vehicle control?
The body of C. elegans is encased in a selectively permeable cuticle that only permits some compounds to be absorbed efficiently through the skin, so the most reliable mechanism for delivering compounds to the worms is through ingestion. Water-soluble compounds easily mix into the nematode growth media and food bacteria and are readily taken up by the worms. Hydrophobic compounds, however, require an organic solvent vehicle such as DMSO which can limit the dosage because DMSO itself can be toxic above a concentration of 0.1%. For these hydrophobic substances, it is important to mix into the food bacteria so that there isn’t any question if it is being ingested by the worms, however mixing hydrophobic compounds into the water rich food bacteria can cause precipitates to form. We have personally observed compounds (such as Resveratrol and Urolithin A) which have a body of literature supporting life extending effects, but due to precipitate formation on the plates, worms become physically injured, shortening their lifespan.
One of the most common drugs used in C. elegans experiments (especially lifespan experiments) is 5-Fluoro-2′-deoxyuridine (FUdR). FUdR blocks nuclear DNA replication by inhibiting thymidylate synthase thereby blocking reproduction in C. elegans and proliferation of the progeny. By moving L4 stage C. elegans to plates containing ~50uM FUdR you are able to maintain a synchronized population of aged nematodes with rare progeny breakthroughs. Several factors such as: the brand used, time since solution/media preparation, solution/media storage environment, the synchronization of your nematode population, strain, worm food source, exposure to heat from agar preparation and developmental stage can influence the response to FUdR and lead to unintended stress.
Figure 1. 5-Fluoro-2′-deoxyuridine (Milipore Sigma, n.d.).
We at InVivo use this FUdR brand at a concentration of 50uM mixed directly with the NGM on an inactivated food source and have good progeny suppression and sterilization of adults for most strains [Image 1]. Mutant strains that are more sensitive to environmental stressors or experiments that require live bacteria can receive lower doses although 50uM is still a good benchmark to start from. FUdR has life extending properties when exposure is at or beyond the L4 stage, however exposing worms to FUdR before the L4 stage can cause early mortality and induce or reinforce any asynchrony seen in the population. Therefore when we work with multiple strains that have different developmental timings, we make sure to check that at least 90% of the population has reached the L4 stage and the worms are looking well synchronized before introducing them to FUdR. It is better to time FUdR exposure too late rather than too early. For lifespan experiments it is more important to completely inhibit progeny because worms that have had the chance to lay viable progeny do not live as long as sterilized adults. In less stringent cases where you simply need to observe an older population of worms, you can do multiple transfers to fresh FUdR plates until they have been rendered unfertile.
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