TITLE: In vitro excystment of Juvenile Fasciola hepatica
AUTHORS: Paul McCusker, Rebecca Armstrong, Duncan Wells, Emily Robb, Paul McVeigh, Aaron Maule, Erin McCammick, Nathan Clarke, Erica Gardiner

[DESCRIPTION]
This protocol describes our excystment protocol for Fasciola hepatica supplied by Ridgeway Research Ltd. F. hepatica metacercariae (also known as mets or cysts) are supplied attached to Visking tubing with both inner and outer walls intact. This protocol builds upon methodology described by McVeigh et al. (2014) and McCusker et al. (2020) and has been tweaked by many lab members over the years. Our thanks to all who have contributed.


[GUIDELINES]
Storage of F. hepatica metacercariae
When mets are received from Ridgeway Research we move sheets from original sheets into 50 mL falcon tubes in fresh RO water. They are then stored at 4 °C until required and the 'sheet is use' is moved into a 15 mLfalcon tube.

Washing watchglasses
Watchglasses are rinsed with 10 % (v/v) bleach before rinsing in RO water and dried out before subsequent use.


[STEPS]
SECTION: Outer metacercariae (met) wall removal
1. Pipette 300 µL 50% chicken serum (CS50) onto large petri dish base and lid, spread with finger.

SECTION: Outer metacercariae (met) wall removal
2. Fill petri dish base with 10 mL RO water.

SECTION: Outer metacercariae (met) wall removal
3. Gently lift rolled-up met sheet out of tube, place in dish base and unwrap the sheet (ensure you know which way mets are facing).
SECTION: Outer metacercariae (met) wall removal
4. Add 100 µL of RO water to petri dish lid to ensure it stays damp. Lay the met sheet onto the lid with mets facing down. NB: avoid bubble formation.

SECTION: Outer metacercariae (met) wall removal
5. Use a scalpel to gently pop required number of mets out of outer wall (assume ~70-75% excystment rate). Outer wall is coloured brown whereas 'popped' mets are translucent. NB. only count viable mets (those with a bilobed appearance following popping).
SECTION: Outer metacercariae (met) wall removal
6. Add 10 mL water to petri dish lid before moving sheet back to petri dish base (mets facing up).

SECTION: Outer metacercariae (met) wall removal
7. Transfer mets from petri dish lid and base to a watchglass using a serum-lined tip.
SECTION: Solution Preparation
8. Make the following solutions and warm to 37 °C:

SECTION: Solution Preparation
8.1. Tube 1 – Dissolve 20 mg sodium tauroglycocholate in2.5 mL Excystment Salt Solution in a 15 mL falcon tube.

SECTION: Solution Preparation
8.2. Tube 2 – Dissolve 20 mg L-cysteine in 2.5 mL 1/20 N HCl in a 15 mL falcon tube.

SECTION: Bleaching mets
9. Swirl watchglass to gather mets in centre. Remove as much water as possible without drying out mets.
SECTION: Bleaching mets
10. Add 1 mL bleach solution to mets and start timer (time varies between sheets/strains - typically ~2 min 30 s)

SECTION: Bleaching mets
10.1. While bleaching is ongoing add 1 mL RO water to a new watchglass and serum line a p100 tip.

SECTION: Bleaching mets
11. As timer approaches 0 swirl watchglass to group mets. As timer hits 0 collect mets in serum-lined p100 tip and move into watchglass with water.
SECTION: Bleaching mets
12. Swirl mets to group, remove as much water as possible and add another 1 mL RO water to wash

SECTION: Bleaching mets
13. Repeat step 12 x5.
SECTION: Bleaching mets
14. After final wash move mets to fresh watchglass using a serum lined p100 tip in 50 µL.

SECTION: Bleaching mets
15. Mix tubes 1 and 2 from step 8 together (some effervesce should be visible). Briefly vortex and ensure that the resulting solution is not cloudy. NB. if cloudy, do not add to worms and remake.
SECTION: Bleaching mets
16. Pour mixed solutions onto mets and place clean watchglass on top to reduce evaporation.
SECTION: Bleaching mets
17. Incubate in a tupperware lunchbox (line base with damp paper towel) for 1 h at 37 °C.

SECTION: Collection of new excysted juveniles (NEJs)
18. Collect NEJs with a p10 and place into pre-warmed RPMI (glass watchglass is best). After collecting excysted worms continue incubation at 37 °C and collect newly excysted NEJs at 20 min intervals until 3 h after addition of excystment solutions.