Fig 1.
Comparison of bleach and filtration techniques to collect embryos.
Bleach synchronization involves dissolving the hypodermis of gravid adults and releasing the embryos. Filtration involves using size-specific filters to separate the embryos from the adults. The figure was created using BioRender.
Fig 2.
Set up of filtration apparatus.
(1) A funnel is used to pour the embryo solution. (2) 40 µm mesh filter to retain adults. (3) 20 µm mesh filter to retain embryos. (4) Connector ring to connect to a vacuum to allow the embryo solution to pass through. (5) A collection tube is used to collect the flow through, which consists of the majority of larvae.
Fig 3.
Collection of embryos for filtration.
(A) A 6 cm OP50-seeded plate with P0 adults and G1 embryos. (B) A 6 cm OP50-seeded plate after the P0 adults have been washed away with M9 buffer. (C) An OP50-seeded plate after the G1 embryos have been scraped off in M9 buffer with a rubber policeman. The figure was created using BioRender.
Fig 4.
Set up of the filtration apparatus.
(1) The G1 embryo solution collected in Fig 3C was poured through the filtration apparatus. (2) The connector ring was connected to a vacuum to allow the solution to pass. (3) After the solution had passed, the filtration apparatus was disassembled. (4) The 20 µm filter was inverted and placed on top of a clean 50 mL labeled tube. (5) A 25% bleach solution was added to the inverted 20 µm filter for 30 seconds to kill any G1 larvae. The figure was created using BioRender.
Fig 5.
A higher number of G1 embryos were recovered from filtration than by bleach synchronization.
Box plot showing G1 embryonic yield per plate obtained by bleach synchronization or filtration protocols. The median is represented by the solid horizontal line with the 75th and 25th quartiles on the top and bottom of the box, respectively. Whiskers extend from the box to the maximum and minimum values that fall within 1.5 times the interquartile range above and below the 75th and 25th percentiles, respectively. The three different colors of the points indicate three experimentalists. A significantly higher number of G1 embryos were collected by filtration (p = 0.00064, Wilcoxon rank-sum test). Eight replicates were tested by each experimentalist for each method.
Fig 6.
Embryos obtained by filtration developed comparably to the embryos obtained by bleach.
The box plot shows the length of L4 animals that developed from G1 embryos collected by bleach or filtration protocols. Median is represented by the solid horizontal line with the 75th and 25th quartiles on the top and bottom of the box, respectively. Whiskers extend from the box to the maximum and minimum values that fall within 1.5 times the interquartile range above and below the 75th and 25th percentiles, respectively. Each point is the summarized measurement of an individual well containing between five and 30 animals. G1 embryos collected by filtration grew into animals with longer lengths compared to lengths of animals that developed from G1 embryos collected using bleach synchronization (p = 0.0025; Wilcoxon rank-sum test). The assay was carried out in three replicates independently by three experimentalists (represented by the three different colored points).
Fig 7.
G1 embryos collected by filtration developed similarly to the G1 embryos obtained by bleach synchronization when exposed to an anthelmintic compound.
Box plots showing normalized animal length for larvae developed from G1 embryos obtained by bleach synchronization or filtration protocols for six C. elegans strains. Median animal lengths of strains exposed to 30 μM albendazole were normalized to the mean of all median animal lengths for the control condition. The median is represented by the solid horizontal line with the 75th and 25th quartiles on the top and bottom of each box, respectively. Whiskers extend from each box to the maximum and minimum values that fall within 1.5 times the interquartile range above and below the 75th and 25th percentiles, respectively. Each point is the summarized measurement of an individual well containing between five and 30 animals. The box plot is faceted by condition (control and drug). The normalized animal length of larvae that developed from G1 embryos collected by filtration is comparable or significantly longer compared to the normalized animal lengths of larvae that developed from G1 embryos collected by bleach, as indicated by the p-values (Wilcoxon rank-sum test) in each facet. The grey background color emphasizes the strains that are sensitive to ABZ. The assay was replicated three times, as indicated by the colors of the points.