Decayment of locomotor activity is a robust readout for detecting early onset of aging. It allows the easy discovery of new genes and the search for compounds to reverse aging. In this experiment we show the application of SMART to measure activity of worms in 35mm Petri dish plates using NGM medium. We introduce the full protocol for motility based lifespan research.
MATERIAL
-NGM-FUdR (50uM – Sigma Aldrich) plates seeded with UV killed E. coli OP50 (Petri Dishes 35x10mm) + Streptomycin 100 µg/ml
-Laboratory film
-Synchronized worms in L4 stage
-Picker
-SMART machine
PROCEDURE
1. Preparing the Test Plates
For ultraviolet killing, 10 μl of OP50 was added to each NGM-FUdR plate and left overnight at 20 °C. Plates were then exposed to ultraviolet light for 30 min
2. Preparing the Worms
– Transfer 25 to 30 synchronized L4 worms from culture plates to NGM-FUdR plates seeded with OP50. Be careful not to damage the agar.
-Seal each plate with laboratory film to avoid the dehydration of the medium.
3. Running the Assay: Measure once a day, for 14 days
– Take out the plate from incubator in order to measure the activity using WMicrotracker SMART.
– Before placing the plate into the equipment, stimulate the worm plate mechanically by taping it 3 times on the bench.
-Proceed to measure the activity for 5 minutes using WMicrotracker SMART.
-Return the plate to the incubator at the desired temperature.
*NOTE: In our experience, 6 days per week is enough to get good data points. The remaining day can be extrapoled using the average between the day before and the day after, without significant differences.
4. Data plot example
