Copas biosort machine

Synchronized young gravid adult hermaphroditic cysl-2p::GFP animals grown on E. coli HT115 were transferred onto E. coli HT115 and P. aeruginosa lawns. P. aeruginosa and E. coli HT115 cultures were grown by inoculating individual bacterial colonies into 2 mL and 10 mL of LB broth, respectively, and growing them for 8–10 hr on a shaker at 37°C. E. coli HT115 cultures were concentrated 10 to 20-fold before seeding on plates. Then, 20 µL of each inoculum was plated onto the center of 3.5-cm-diameter SK plates. For full lawns of P. aeruginosa, 20 µL of inoculum was spread to completely cover the surface of 3.5-cm-diameter SK plates. The plates were incubated at 37°C for 12 hr and then cooled to room temperature for at least 30 min before seeding with synchronized young gravid adult hermaphroditic cysl-2p::GFP animals. The COPAS Biosort machine (Union Biometrica) was used to measure the time of flight (length) and fluorescence of individual worms. At least 100 worms were measured for each condition.

Synchronized young adult glp-4(bn2) worms were washed from NGM plates seeded with RNAi-expressing E. coli HT115 into a 15 mL conical tube and rinsed three times. Worm size was determined via flow vermimetry by using COPAS Biosort machine (Union Biometrica).

Spore levels were quantified as described (Szumowski et al. 2014 (link)). Briefly, 500 synchronized N2 L1s were grown on RNAi plates for 24 hr at 25°. These animals were then washed off plates using M9, and transferred to a fresh 6-cm RNAi plate seeded with RNAi bacteria and 2 × 10⁶N. parisii spores, then grown at 25°. At 40 hpi, infected adult animals were washed off of the plates using M9, and then fixed in acetone. Fixed animals were washed three times with PBS containing 0.1% Tween-20 (PBS-T), before transferring to a 96-well plate for sorting with the Union Biometrica COPAS Biosort machine to dispense 50 animals per well into a new 96-well plate. Wells containing 50 infected animals in 50 µl of water were then mixed with 150 µl of lysis buffer containing PBS with 2% SDS, 0.01% 2-mercaptoethanol, and 4 µg/ml DY96; lysis was monitored microscopically, and complete lysis was typically achieved after 2 hr incubation at room temperature. Spores were then counted by visualizing DY96 stained spores on a hemocytometer.
Spore levels were quantified in mutant worms identically as described above, except that RNAi bacteria with an empty RNAi vector was used (HT115E. coli with L4440 plasmid) as the food source. All experiments were performed in independent triplicates and analyzed for statistical significance using ANOVA and GraphPad software (Prism).

Synchronized L1 stage animals were grown at 20°C to the L4 stage. The COPAS Biosort machine (Union Biometrica) was used to measure the time of flight (as a measure of length) and fluorescence of individual worms. At least 100 worms were measured for each strain, and all experiments were performed in biological triplicate.

N. parisii spores were prepared as previously described [82 ]. Spores (1 million per plate) were mixed with food and L1 (Fig 6C) or L4 (Fig 6D) synchronized animals. To reach the L4 stage, worms were grown on NGM plates for 72 h (pals-17(*) mutants) or 48 h (all other strains) at 20 °C. During infection, animals were incubated at 25 °C for 30 h. Animals were collected and fixed in 4% paraformaldehyde for 30 min. Fixed worms were stained at 46 °C overnight using MicroB FISH probe conjugated to the red Cal Fluor 610 fluorophore (Biosearch Technologies), targeting microsporidia ribosomal RNA [81 (link),82 ]. N. parisii pathogen load was measured with the COPAS Biosort machine (Union Biometrica). Data analysis was performed using GraphPad Prism 9. Raw data are shown in S1 Table.

N. parisii spores were isolated as previously described [25 (link)]. 1200 synchronized L1 worms were mixed with 5x10⁶N. parisii spores, 25 μl 10X concentrated OP50-1 bacteria and M9 to bring the total volume to 300 μl. This mixture was then plated on room temperature unseeded 6 cm NGM plates, allowed to dry and then incubated at 25°C for 3 hours or 30 hours. Three plates were used per genotype. Animals were fixed in 4% paraformaldehyde and then stained using a FISH probe specific to N. parisii ribosomal RNA conjugated to Cal Fluor 610 dye (Biosearch Technologies). For the 3 hpi timepoint, pathogen load was determined by counting sporoplasms per worms using 40x objective on a Zeiss AxioImager M1 microscope. For each replicate, 75 animals per genotype were quantified. Three experimental replicates were performed. For the 30 hpi timepoint, pathogen load was quantified using the COPAS Biosort machine (Union Biometrica). The N. parisii FISH signal for each worm was normalized to the length of the worm using time-of-flight measurements. For each replicate, 100 animals per genotype were quantified.

For Western blot measurements of protein levels, animals were washed off NGM plates with M9 buffer, and then washed once with PBS with 0.1% Tween 20 to remove bacteria. Samples were then resuspended in sample buffer with 1% SDS and 50 mM DTT and boiled at 95°C for 10 minutes. Lysates were then run on a 4–20% gradient SDS-PAGE gel (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). The blots were stained with Ponseau S stain (Sigma-Aldrich) and imaged for total protein before staining with an anti-GFP antibody made in rabbits (a gift from Arshad Desai and Karen Oegema labs at UC San Diego) diluted at 1:5000 overnight at 4°C, then staining with an antirabbit HRP antibody at 1:10000 for 45 minutes at room temperature. The blots were treated with ECL reagent (Amersham GE Healthcare Life Sciences) and imaged on a Bio-Rad ChemiDoc. For measurements of GFP protein levels in live animals, uninfected and N. ironsii infected animals were collected 15 hour post-inoculation (hpi) and measured on a COPAS Biosort machine (Union Biometrica) for size (time-of-flight, TOF) and GFP (green). More than 1000 animals were measured per sample. Animals expressing transgenic GFP were distinguished from non-expressing animals by measuring GFP levels in wild type N2 animals and only analyzing GFP levels in animals above this threshold.