This method can help answer key questions about the cell cycle and germ line stem cell population dynamics in C.elegans. The main advantages of this technique are that it requires no transgenes, it is compatible with immunofluorescent staining, and it can be modified to study cells under many different conditions. I was very excited when I first saw this method being used in the Scheda lab.
Though this method can provide insight into the C.elegans cell cycle, it can also be applied to broader questions about aging, nutrition, or altered genotypes. Generally labs new to this method may struggle with the initial idiodisprepation or C.elegans hematoxylin staining. Begin by using sterile technique to add 200 microliters of 10 millimolar EdU in 100 milliliters of M9 buffer and the appropriate supplements to four milliliters of freshly grown overnight MG 1693 E.coli culture.
Making idiodiscious requires a delicate balance between EdU and thymidine supplementation. As the amount of thymidine must be sufficient for the E coli to grow well, while the amount of EdU must be sufficient for the robust shield against leveling. After no more than 24 hours at 37 degrees Celsius and 200 RPM, use sterile technique to split the culture between two to four sterile 50 milliliter conical tubes for centrifugation.
Resuspend the pellatin four milliliters of fresh M9 buffer and use the same pipette to apply about 8 drops of EdU labeled E Coli MG1693 solution to the center of individual room temperature 60 milliliter M9 A guard petri dishes. To feed the EdU labeled bacteria to the nematodes, use fresh PBS to wash the C.Elegans from a nematode growth medium dish into a 1.5 milliliter tube and allow the animals to settle briefly by gravity. Wash the animals one to two times with one milliliter of PBS and use a glass pasteur pipette to transfer the nematodes onto the center of the EdU lawn in a tiny drop of PBS.
Wait a few minutes for the liquid to be absorbed and incubate the culture for at least 30 minutes at 20 degrees Celsius according to the experimental protocol. At the end of the incubation, use two milliliters of PBS to flush the worms from the EdU culture plate into a glass dissecting dish. After dissection and fixation of the nematode gonads as previously described, rinse a small one milliliter borosilicate glass tube and a long glass pasteur pipette with PBS supplemented with 0.1%tween 20 and use the pipette to transfer the fixed gonads to the tube in a small amount of PBS tween.
Collect the gonads by centrifugation and use a long, drawn out glass pasture pipette to remove as much supernatent as possible without disturbing the gonad pellet. For EdU detection, add 100 microLiters of click EdU cocktail to the gonads and cover the tube with laboratory film for a 30 to 60 minute incubation at room temperature. At the end of the incubation, wash the gonads one time with 100 microLiters of reaction rinse buffer, and four times with one milliliter of PBS tween.
After the last wash, add 25 microLiters of anti-fade mounting medium supplemented with DAPI to the sample. While the medium is settling over the gonads, place a large 2.5%agarose pad onto a standard glass microscope slide. Then flatten with a second slide.
Next, use a long glass pasteur pipette and keep all of the liquid and gonads in the narrow tip of the pipette to minimize sample loss when transferring the gonads to the pad. Using an eyelash glued to a toothpick, distribute the gonads over the agarose pad and remove any dust particles. Then slowly lower a rectangular glass cover slip onto the gonads, taking care to avoid air bubbles, and removing any excess solution with a lab tissue.
Counting cells in three dimensions reliably takes some practice. Using Fiji's cell counter plug-in and a script like marks the cells to remove any duplicates helps make the counts reproducible. After allowing the cover slip to settle overnight, use the cell counter plug-in and Fiji to manually count each nucleus, labeling each individual nucleus according to the presence or absence of phosphohistone three EdU or pregenetor zone cell labeling.
EdU signal co-localizes with DAPI signal. In some nuclei, the EdU signal covers all of the chromosomes, while in other nuclei, the EdU signal localizes to one to two bright punkta, likely on the X-chromosome, which replicates late in the S-phase. EdU signal from a successful 30 minute labeling localizes to approximately half of the nuclei in the pregenetor zone.
While the technique works consistently in wild type young adult animals, a significant fraction of mated 5 day old hermaphrodites fails to label in a 30 minute EdU pulse. The duration of G2 can be estimated by analyzing the percentage of nuclei in the M-phase that are EdU positive over the time course, allowing calculation of the median and maximum duration of G2.The duration of G2 and G1 can be estimated from the percentage of all of the pregenetor zone nuclei that are EdU positive, allowing calculation of the maximum duration of the combined phases. While attempting this procedure, it's important to remember to use the same batch of EdU dishes to minimize inter-experimental variability.
Following this procedure, other methods like staining with additional antibodies can be performed or answer additional questions about the cell cycle, cell fate, or signaling pathways. Don't forget that working with nucleicide analogs and parafermeldahyde can be hazardous, and that precautions, such as wearing nitrol gloves, should always be taken while performing this procedure.
