In tissue culture hood, prepare three petri dishes with 1x DPBS+2% anti-anti, one with 70% ethanol, and one clean petri dish arranged in the following order:

Decant the livers into the first petri dish using ethanol-cleaned tweezers to submerge and continuously move them for 1 minute to ensure complete saturation of the tissue

Repeat step 3 for each petri dish with the exception of the 70% ethanol dish which should be submerged and rinsed for less than 10 seconds

Place all livers into a clean petri dish and using a sterile scalpel finely mince them

Transfer the minced livers to a 50ml falcon tube, re-suspend in 40ml of 1x DPBS + 2% anti-anti and label with necessary identifying information

Weigh out 0.05g of collagenase into a labelled 15ml falcon tube and add 10ml of dH₂0. Mix well (The amount of collagenase to prepare depends on the number of livers to be processed – collagenase is always prepared fresh)

Add 5ml of 0.5% collagenase solution to the liver suspension and mix well

• Optional: Add to the mix 500 ul of polymixin B (100K Units) (Sigma-Aldrich, P4932-1MU), a gram negative bactericidal antibiotic that reduce LPS contamination in the egg prep, in particular when SEA will be prepared from eggs and immunological studies or co-culture with cells, will be performed

Wrap securely in parafilm and secure the tube horizontally in a 37°C rocker overnight

The following day, centrifuge the liver suspension tube at 400g for 5 min (acceleration and deceleration 9)

Aspirate the supernatant and re-suspend in 50ml of 1x DPBS + 2% anti-anti.

Repeat steps 9-10 three more times

After the final aspiration, re-suspend the pellet in 25ml of 1x DPBS + 2% anti-anti

Using a 50ml stripette, pass the suspension through the 250uM sieve into a 1L sterile beaker

Pass this filtrate through the 150uM sieve into a second 1L sterile beaker

Wash the beaker with 5ml of 1x DPBS + 2% anti-anti to collect any remaining eggs and add this to the filtrate by passing through the 150uM sieve

Decant the filtrate into a 50ml falcon tube and centrifuge (400g, 5 minutes, acceleration and deceleration 9)

Aspirate the supernatant and resuspend in 10ml 1x DPBS + 2% anti-anti

Prepare a Percoll gradient (one percoll gradient per 5 livers):

Prepare a 0.25M sucrose solution (4.27g sucrose + 50ml diH₂O) and filter the solution through 0.22um syringe filter

In 50ml falcon tube mix 8ml Percoll and 32ml of the 0.25M sucrose solution. Invert 5 times

Very carefully pipette the resuspended eggs onto the surface of the Percoll gradient around the circumference to create a defined layer

Centrifuge the gradient at 800g for 10 minutes (acceleration 2 and deceleration 1)

Aspirate the supernatant and re-suspend in 10ml of 1x DPBS + 2% anti-anti. Transfer to a 15ml falcon tube

Centrifuge at 400g for 5 min (acceleration and deceleration 9)

Repeat steps 21-22 two more times

IMPORTANT. Check the eggs under microscope, if the prep is still ‘dirty’ or ‘contaminated with liver debris’ proceed with a second percoll gradient

Resuspend the pellet in 10ml 1x DPBS + 2% anti-anti and count 12 aliquots of 5µl to estimate total number of collected eggs

Centrifuge eggs in falcon tube at 400g for 5 mins (acceleration and deceleration 9)

Resuspend eggs in adult media (DMEM + 10% FBS + 2% anti-anti) and transfer to 6 well plates (5-6ml of media per well)

Keep eggs in culture at 37°C and 5% CO₂

• Eggs can be kept in culture for up to ~10 days and retain the ‘hatchability’ however, the hatching rate will drop over time

Centrifuge eggs in falcon tube at 400g for 5 mins (acceleration and deceleration 9)

In the culture hood, aspirate supernatant and re-suspend in 6ml diH₂O

Aliquot 1ml each in a 24 well plate

• It is important to use 24 well plate given the miracidia get more diluted in 12 or 6 well plate and more egg shells are picked up when collecting the miracidia)

Rinse the original falcon tube with 6 ml of water and distribute 1 ml to each well containing 1ml of eggs (i.e. the eggs will be in 2ml of water)

Place under light for hatching

At 30-40 min intervals over ~3 hrs, gently remove the top 1ml of water containing the miracidia into a 50ml falcon tube and top up the wells with 1ml of diH₂O

Count miracidia and proceed with snail infections dx.doi.org/10.17504/protocols.io.36wgqjkkxvk5/v1 or sporocyst transformation

Place the tube containing miracidia on ice for ~20 min

Centrifuge at 800g for 15min (acceleration and deceleration 9)

Quickly aspirate the water and resuspend the pellet of miracidia in complete sporocyst media

• Sporocysts can be kept in culture at 28°C with malaria gas (90-92% N, 5% CO₂, 3-5% O₂) in a sealed container changing the media once to twice a week
• IMPORTANT. To avoid contamination always replace the media with fresh complete media the day after transformation