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 ARENA to measure activity of worms in 24well plates using liquid medium, and we present the full protocol for motility based lifespan research.
– Synchronized L4 worms (N2 strain).
– Complete S Basal Medium (See recipe below)
– Streptomycin stock 100 mg/ml.
– Kanamycin stock 100 mg/ml.
– Amphotericin B stock 250 µg/ml.
– OP50 feeding bacteria in S-complete media 100 mg/ml.
– WMicrotracker ARENA device.
Sterile conditions are need by using flow cabinet or flame.
We recommend perform at least 4 technical replicates and 2 biological replicates.
DAY 0_ Passing L4 worms to 24-well plate
- Collect the L4 worms from the NGM agar plates by washing the plates with Complete S Basal medium.
- Adjust the final volume so that worm concentration in the suspension is approximately 150 worms/ml (75w/well/500µl).
- Add streptomycin (to a final 200 μg/mL), kanamycin (20 μg/mL) and the antifungal drug Amphotericin B (final concentration 2.5 μg/mL) to avoid contamination.
- Add an appropriate volume of feeding bacteria in S-complete media to a final concentration of 2,5 mg/mL.
- Add FUdR to a final concentration of 50 μM.
- Mix the worm suspension thoroughly by inverting the tube several times.
- Disperse 500 μL of the suspension in each well of a 24-well plate.
- Seal the plate with a transparent adhesive sealer, to avoid contamination and evaporation of the samples.
- Put the plate in the incubator at the desired temperature with agitation (100 rpm).
DAY 1 to 20. _ Measure locomotor activity once a day.
- Before setting the plate into the WMicrotracker ARENA, make sure there is no condensed water on the plate lid. If necessary, dry the plate lid maintaining sterile conditions.
- Shake the microplate of worms gently by hand or stimulate it using high intensity blue light to induce animal movement.
- Record the activity of the plate with worms using wMicrotracker ARENA for 30 min at the same temperature of the incubator.
To prevent starvation of worms, feeding bacteria need to be added.
– After recording the activity of the worms using wMicrotracker ARENA, remove the sealer from the micro-plate maintaining sterile conditions.
– Add 5 μL of bacterial suspension in each well and seal the plate again.
– Shake the culture gently by hand.
– Return it into the incubator.
Acquired data was analyzed and plotted using MSExcel.
The locomotion activity of N2 worms was evaluated during 17 days. On day 0, L4 larvae stage N2 worms were transferred to S Complete Medium with 50 µM FudR in a 24well-plate. The worms were allowed to grow at 24ºC. The locomotor activity was recorded at 24ºC during 30 min. Data represents the mean activity recorded each date of 6 replicates per group.
Additional RECIPES (from Ian Hope book)
– S-basal medium:
For 1 L, add:
5.9 g NaCl,
50 mL of 1 M Potassium Phosphate Buffer pH 6.0
Adjust the volume with ddH2O up to 1 L and autoclave.
Let the medium cool down and add 1 mL of 5 mg/mL cholesterol (dissolved in ethanol).
– S-complete medium:
For 1 L, add:
977 mL S-basal medium,
10 mL 1 M Potassium citrate pH 6 (sterile)
10 mL Trace metal solution (sterile)
3 mL 1 M CaCl 2 (sterile)
3 mL 1 M MgSO 4 (sterile).
Use sterile technique, do not autoclave.