Biosorter

Positive controls for all screens are png-1 mutants+DMSO (vehicle control) and the negative controls are png-1 mutants+DMSO+205 nM bortezomib. Both drug libraries were screened in triplicate with controls on each 384-well drug-screening plate. The final DMSO concentration in each well was 0.27%. Worms tolerated up to 1% DMSO before showing signs of toxicity. Using the Echo550 liquid handler (Labcyte Inc.), bortezomib was acoustically dispensed into the destination plates the day before the worm larvae sort. In total, 5 µl of HB101 bacteria were dispensed into 384-well plates containing S-medium (prepared in-house) in each well. Using the BioSorter (Union Biometrica), 15 L1 png-1 mutant larvae were sorted into each well, and plates were incubated for 5 days at 20°C while shaking. After 5 days of benchtop incubation, 15 µl of 8 mM sodium azide was added to each well to immobilize worms prior to imaging in a custom worm imager. Finally, automated image processing was run on each plate.

Larval biosorting was completed with a Biosorter (Union Biometrica Inc) with or without an associated Large Particle Handler (Union Biometrica Inc). When sampling from a 96-well plate, larvae were arrayed with four larvae/well, and were exposed to 0.2 mg/ml tricaine immediately before sampling, to ensure linear passage of the larvae through the tubing. For these experiments, the unit was fitted with a 500 μM fluidics and optics core assembly (FOCA), 1000 μM fluid handling tubing, and a 488 nm laser. Although data were collected on all channels simultaneously, PH green fluorescence was examined in YO-PRO-1 studies, and overall green fluorescence was assessed in the GFR studies.

To quantify fluorescent reporters, flow cytometry using a Union Biometrica bioSorter (cat. no. 250-5000-000) was done as previously described (53 ). Briefly, staged worms were washed off plates using M9, allowed to settle by gravity, and washed once with M9 to separate from eggs. The signal was collected for time of flight (TOF; length) and extinction (thickness) of animals, along with the GFP and red fluorescent protein (RFP). Data were collected gating for size (TOF and extinction) to exclude eggs. Data are represented as an integrated intensity of fluorescence normalized to the size of the animal using the integrated GFP output and dividing by the extinction and TOF. All data that exceed the measurement capacity of the photomultiplier tube (PMT), calculated as a signal of 65,355, are considered saturated and are censored from the calculation. For spatial profiles, the complete profiles were extracted, and worms were aligned according to their myo-2p::tdtomato (red head) signal using MATLAB (MathWorks) and binned into 100 bins to account for differences in animal length (n > 50). Then, the average profile and SEM were calculated on binned profiles. For hsp-6p::GFP worms, which do not harbor a myo-2p::tdtomato coinjection marker, the profiles were aligned according to the GFP signal, with the highest peak of signal defined as the posterior intestine.

To quantify fluorescent reporters, flow cytometry using a Union Biometrica bioSorter (cat. no. 250-5000-000) was done as previously described (53 ). Briefly, staged worms were washed off plates using M9, allowed to settle by gravity, and washed once with M9 to separate from eggs. The signal was collected for time of flight (TOF; length) and extinction (thickness) of animals, along with the GFP and red fluorescent protein (RFP). Data were collected gating for size (TOF and extinction) to exclude eggs. Data are represented as an integrated intensity of fluorescence normalized to the size of the animal using the integrated GFP output and dividing by the extinction and TOF. All data that exceed the measurement capacity of the photomultiplier tube (PMT), calculated as a signal of 65,355, are considered saturated and are censored from the calculation. For spatial profiles, the complete profiles were extracted, and worms were aligned according to their myo-2p::tdtomato (red head) signal using MATLAB (MathWorks) and binned into 100 bins to account for differences in animal length (n > 50). Then, the average profile and SEM were calculated on binned profiles. For hsp-6p::GFP worms, which do not harbor a myo-2p::tdtomato coinjection marker, the profiles were aligned according to the GFP signal, with the highest peak of signal defined as the posterior intestine.

MAPs treated as required were fixed as above, washed twice in PBS for 10 min at 4 °C, and transferred to PBS with 1 percent FBS. MAPs were sorted at 561 nm on Biosorter (Union Biometrica) equipped with a FP-1000 nozzle.

Synchronized populations of worms were grown on control (L4440), dld-1(RNAi) (LLC1.3), or 1:20 dld-1(RNAi). Each population was monitored for development at the L4 stage and days 1, 5, and 10 of adulthood. At the L4 stage, worms from each population were transferred onto plates containing 50 μM fluorodeoxyuridine to prevent the birth of progeny for analysis at adult days 1, 5, and 10. On the appropriate days, L4 larvae to day 10 adults were collected from the plates and washed in 4 to 5 mL S basal in a 50 mL conical tube (obtained from Children’s Hospital of Philadelphia). Flow cytometry analyses of animal length were performed using a BioSorter (Union Biometrica), which measures the relative axial length of an object by an axial light loss detector, where time of flight (TOF) indicates animal length (63 (link)). TOF measurements were collected for approximately 100–300 worms per sample, with 4 biological replicates tested per condition.