AMPK Methods and Protocols

3 Methods

3.1 Effects
on the Parental (P 0 )
Generation

3.1.1 Alkaline
Hypochlorite Treatment
and L1 Survival

1. Defrost strains and let them grow without any period of star-
   vation for at least three generations at 20C prior to starting
   the experiment.


2. Collect gravid adults from three 6 cm NGM (Nematode
   Growth Media) plates seeded with E. coli OP50 bacterial
   lawns by washing plates with M9 or water and transferring
   the solution to a 15 mL polystyrene conical test tube (see
   Note 2).


3. Pellet the animals by centrifuging at low speed for 1 min
   (~1500 rpm on a standard tabletop centrifuge, 400g)at
   room temperature, and gently aspirate the supernatant taking
   care not to remove the animals in the pellet.


4. Perform 1–3 washes by filling the tube with M9 and repeating
   the aspiration step until the wash buffer is clear of bacteria.


5. Add 1 volume of alkaline hypochlorite solution for every vol-
   ume of larvae collected in M9 buffer. Vortex and invert the
   tubes during the incubation period until the cuticle of the adult
   animals is fully dissolved and there are no traces of animal
   debris (seeNote 3).


6. When there is no visible remaining debris in the tube, stop the
   reaction by dilution by adding M9 buffer to fill the tube (see
   Note 4).


7. Speed is critical, so quickly centrifuge for 1 min at 400g, and
   discard the supernatant. Repeat this twice to remove all the
   diluted alkaline hypochlorite solution, carefully aspirating the
   wash buffer at each step to avoid taking up the isolated
   embryos.


8. Add 2–3 mL of sterile M9 buffer to the pellet, and incubate
   overnight at the desired temperature with gentle agitation.


9. All embryos should have hatched by the next morning, and
   only live L1 larvae should be present in the buffer.


10. Adjust the titer so that it is 6–10 L1 larvae/μL. L1 larvae are
    very fragile and should be manipulated with care at all times.
    Some animals may die due to the procedure, and this number
    can be determined after the L1 larvae crawl out of the drop
    onto the bacteria (seeNote 5).


11. The emergent L1 larvae can be maintained in this starved state
    (L1 diapause) for varying durations to test the effects of star-
    vation on various parameters (seeNote 6).


12. After the selected durations in the diapause, animals can be
    aliquoted onto plates containing OP50 to initiate

Quantifying Starvation-inducible Transgenerational Defects 569