Synergy high speed cell sorter

Synchronized transgenic young adults in which the ADL neurons were specifically labelled using srh-220p:mKate2 were washed 5× in M9 buffer to remove bacteria and then dissociated as described (Beets et al., 2020 (link); Kaletsky et al., 2018 (link)). Briefly, animals were incubated for 6.5 min in Lysis buffer (200 mM Dithiothreitol (DTT), 0.25% Sodium dodecyl sulfate (SDS), 20 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3% sucrose) washed 5× in M9 buffer, resuspended in 500 μl of 20 mg/μl Pronase in water, and pipetted up and down for 12 min at room temperature. The reaction was stopped by adding 250 μl of 2% FBS in PBS. Cells were filtered through a 5-μm syringe filter to remove clumps. mKate(+) cells were sorted using a Synergy High Speed Cell Sorter (Sony Biotechnology) with gates set using a negative control prepared in parallel from dissociated unlabelled N2 animals. Positive cells were collected into 10 μl of Triton X-100 0.2% (vol/vol) supplemented with 2 U/ml RNase inhibitors. Between 700 and 3000 cells were collected for each biological replicate.

Synchronized young adult hermaphrodites with GFP-labeled BAG neurons (expressing a flp-17p::gfp transgene) were acutely dissociated as described (Kaletsky et al., 2016 (link)). Synchronized adult worms were washed with M9 buffer to remove excess bacteria. The pellet (∼250 μl) was washed with 500 μl lysis buffer (200 mM DTT, 0.25% SDS, 20 mM HEPES pH 8.0, 3% sucrose) and resuspended in 750 μl lysis buffer. Worms were incubated in lysis buffer for 6.5 min at room temperature. The pellet was washed 5 times with M9 and resuspended in 20 mg/ml pronase from Streptomyces griseus (Roche). Worms were pipetted up and down for 12 min at room temperature; then ice-cold PBS buffer containing 2% fetal bovine serum (GIBCO) was added. Cell suspensions were passed over a 5 μm syringe filter (Millipore). The filtered cells were diluted in PBS and sorted using a Sony Biotechnology Synergy High Speed Cell Sorter. Gates for detection were set by comparison to npr-1 cell suspensions prepared on the same day alongside the experimental samples. Positive fluorescent events were sorted directly into Eppendorf tubes containing 10 μL of 0.2% (vol/vol) Triton X-100 and 2 U μl^-1 RNase inhibitor. Six biological replicates were prepared for each genotype, i.e., npr-1(ad609) and arcp-1(db1082); npr-1(ad609) animals. For each replicate sample, approximately 4,000 GFP positive events were collected.