Synchronized populations

To achieve tightly synchronized populations without potential biological artifacts introduced by a bleach or starvation synchronization protocol, we utilized the NemaSync C. elegans synchronizer model 5000 (InVivo Biosystems). Mixed-stage animals were grown to high density on 12–20 10 mm nematode growth medium plates seeded with Escherichia coli OP50. Animals were then washed off in M9 buffer and added to the stabilization filter, allowing gravid adults to be separated from all other stages. Adult animals were collected from the filter and pipetted onto the harvest filter. Recently hatched L1 larvae collected from 15-min windows were then re-plated onto OP50-seeded plates and grown at 20°C for precisely the desired time. All reported times for time-lapse imaging refer to time post-plating of synchronized L1 animals.