Sex and culture duration differences in Ascaridia galli Extracellular Vesicles
Description
To determine how to obtain the highest amount of EVs shed by A. galli, several kinetic experiments were conducted. Cleaned and sexed worms were grouped according to size. For both females and males, medium-sized worms (females: 5.7-6.8 cm; males: 3.7-4.8 cm) produced the most particles per mL, indicating that EV production depends on life stage. The amount of EVs shed by A. galli was relatively stable across replicates, though the optimal culture duration for maximal EV yield differed between sexes. Female worms produced the highest average particle yield after 24 hours of culture, with no significant difference compared to 48 hours. Male worms shed the highest number of EVs after 40 hours, but again, no significant difference was found between timepoints. No morphological differences were observed between EV fractions from different timepoints or worm sexes. Following size-exclusion chromatography, EVs from females were mainly detected in fractions 5-8, with the highest yield in fraction 6. In contrast, male worms produced significantly fewer EVs overall, and none were detected in fraction 8. The size distribution of EVs did not differ between sexes, with most particles ranging from 100 to 200 nm.
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Steps to reproduce
Collection and Initial Handling 1. Collect adult A. galli worms from the small intestines of hens immediately after dissection. 2. Transfer worms into 0.9% NaCl solution and maintain at room temperature during transport. 3. Upon arrival in the lab, wash worms three to five times with PBS at room temperature. Sorting and Preparation 4. Separate worms by sex and categorize by size (small, medium, large). 5. Wash worms three times with EV-free RPMI 1640 medium supplemented with vesicle-depleted fetal bovine serum. In Vitro Culture 6. Transfer 20 worms into T75 culture flasks containing 30 mL of EV-free RPMI medium. 7. Incubate worms at room temperature, collecting spent medium (containing EVs) at 18 h, 24 h, 40 h, and 48 h. 8. Centrifuge sequentially to remove eggs, cells, and debris: 300 × g for 10 min (remove eggs), 400 × g, 4,000 × g, and 10,000 × g for 10 min each at 4 °C 9. Filter the supernatant through a 0.45 µm polyethersulfone syringe filter. 10. Store collected medium at –80 °C until further EV enrichment. Enrichment of EVs with Size Exclusion Chromatography 11. Load the concentrated sample onto a 10 cm Sepharose 4 Fast Flow column (PBS as eluent). 12. Collect 20 fractions (500 µL each). 13. Identify EV-rich fractions (typically fractions 5–7) based on nanoparticle tracking analysis (NTA) and Bradford assay. Nanoparticle Tracking Analysis (NTA) 14. Measure particle size and concentration using a ZetaView PMX 110 instrument. 15. Perform auto-alignment with 100 nm polystyrene standards. 16. Record data in scatter mode (camera sensitivity 85, shutter 70, frame rate 30 fps, 3 cycles × 11 frames).
Institutions
- Aarhus Universitet Faculty of Natural Sciences
- Eesti Maaulikool
Categories
Funders
- HORIZONGrant ID: No 101079349
- HORIZON 2020Grant ID: No 857418
- Horizon EuropeGrant ID: Project 101136346