While other solid culture methods exist, this is the first solid culture system that allows automated longitudinal tracking of individual animal life and health span and longitudinal imaging of fluorescent biomarkers in one system. This protocol provides comparable results to standard C.elegans experiments on solid NGM is compatible with both aversive interventions and direct live fluorescence imaging. This system will be invaluable for aging research, especially regarding long-term exposure to environmental stressors and observing gene and protein dynamics over extended periods.
Avoiding contamination and getting consistent drying across the wells are both challenging. We highlighted key points in the written protocol to minimize these issues. To begin, grind 500 milligrams of solid palmitic acid in a mortar and pestle.
Combine it with 15 milliliters of TWEEN 20 in a 50 milliliter conical vial, and mix by vortexing to dissolve the crystals. Add 17.5 milliliters of 100%ethanol, and vortex the solution to dissolve as much palmitic acid as possible. Then add 17.5 milliliters of ultrapure water, and vortex vigorously.
Gently heat the solution in a bead bath at 80 degrees Celsius until the palmitic acid dissolves completely. Reheat the bead bath to 80 degrees Celsius. Immediately before use, aliquot one milliliter of the palmitic acid working solution per Microtray into a separate sterile container, and add four microliters of nystatin per one milliliter of palmitic acid solution before heating.
Now spray inside and outside the Microtray with ethanol to saturation, and shake off the excess ethanol. To sterilize the Microtray, place the plate and lid separately in the UV crosslinker. After 20 minutes, flip the plate and lid.
Next, place the 15 milliliter conical vial with the palmitic acid working solution in the bead bath, and allow any visible crystals to dissolve completely before use. Warm up the Microtray by placing it on the ledge surrounding the bead bath with the lids on for two minutes. Remove the Microtray lid, and slowly add 200 microliters of palmitic acid working solution across the back wall of the Microtray.
Replace the lids, and let the Microtray sit on the bead bath for two to three minutes to allow the palmitic acid solution to settle across the bottom of the Microtray. Rotate the Microtray 180 degrees, and add the palmitic acid working solution as demonstrated earlier. Prepare lmNGM by adding the components described in the manuscript.
Then pipette 20 microliters of lmNGM into each Microtray placed on the bench. Be sure to cover the Microtray when refilling the pipette to minimize contamination. To seed the Microtray wells with bacterial food, centrifuge a culture grown overnight at 3, 500 G for 20 minutes.
Remove the supernatant, and resuspend the bacteria culture at a tenfold concentration with 85 millimolar sodium chloride solution. Then carefully pipet five microliters of tenfold concentrated bacterial culture onto the surface of the lmNGM in each well. Avoid allowing the bacteria culture to run over the side of the well and into the palmitic acid, as this will attract worms to leave the well.
Place the sterile 3D printed insert into a sterile single-well plate. Place the Microtray into the 3D printed adapter in the single-well plate. Liberally dispense water crystals on top of the 3D printed insert between the outer well of the Microtray and the inner wall of the single-well plate.
If worms are not being added immediately, seal the Microtray to use the next day. Close the tray lid, and use a single piece of Parafilm to wrap all four sides to seal the Microtray. Repeat the sealing step with Parafilm two more times for three layers.
Add one worm per well once they've reach the desired life stage. Add 40 microliters of detergent solution to the lid of a single-well plate, and use a task wipe to coat the lid evenly. Use additional task wipes to spread the detergent solution until vision through the lid is not distorted.
Stretch the first piece of Parafilm slightly to cover two sides of the tray. Wipe the top of the tray with a task wipe, and damp it with 70%ethanol to remove any fingerprints. Measure the lifespan by gently tapping the side or top of the plate, or shining a bright blue light on the plate, and observing each animal.
Score an animal dead if it does not move within a few seconds. Repeat this task every one to three days until all worms have died. Perform fluorescent imaging on single wells.
Exposure to copper sulfate and dithiothreitol significantly reduced worm lifespan and health span. In contrast, copper sulfate decreased the proportion of life spent in good health, while dithiothreitol did not. Copper sulfate reduced average activity across individuals within the population to a greater extent than dithiothreitol at all ages.
The population average of the individual animal area under the lifetime activity curve is substantially impaired by copper sulfate or dithiothreitol. Cumulative activity for individual animals up to day 10 correlates with their lifespan for dithiothreitol challenged animals, but not for untreated controls or copper sulfate challenged animals. A drastic increase in KYNU-1 expression is observed between days three and eight of adulthood, and there is a progressive decline with age after that.
Cumulative KYNU-1 mScarlet expression through day 14 of adulthood correlates with animal lifespan. Since KYNU-1 expression is dynamic throughout a lifespan, we compared KYNU-1 expression at different ages to overall survival across individual animals. At no single time point was KYNU-1 expression significantly correlated with survival.
When attempting this procedure, the most important steps are addition of the palmitic acid, pipetting the agar, and introducing the worms. This technique allows longitudinal tracking of lifespan and health span, as well as fluorescent biomarkers in individual worms in a way that is directly comparable to NGM assays.
