Schistosoma mansoni cercariae transformation (with needle)

Shed cercariae from snails in a 6-well plate (see protocol "Schistosoma mansoni cercariae shedding"). The snails can be shed for up to 2 hrs by collecting cercariae and replacing with fresh water every 30 min

Using a sterile transfer pipette, dispense cercariae into an autoclaved 50ml beaker

Fill the beaker containing cercariae to 45-50ml with diH₂O and incubate for 0h 30m 0s``On ice

Carefully transfer the cercariae to a 50ml Falcon tube and centrifuge at 500x g,4°C

Discard the supernatant and add 5ml 1x PBS + 2% Anti-Anti to the pellet. Gently resuspend by flicking the tube (avoid using pipette because the cercariae are quite sticky and get lost)

Add 45ml 1x PBS + 2% Anti-Anti and centrifuge at 500x g,4°C

Repeat this steps 6-7 once more with 1x PBS + 2% Anti-Anti and then twice with somule wash

During the centrifugations in Steps 7 and 8, prepare 2 percoll gradient tubes (2 tube for less than ~150K cercs, 4 tubes every ~150K cercs)

Following centrifugation of the cercariae, remove the supernatant and add 5ml of somule wash per percoll gradient you will use

Vortex for 1 min at max speed. This will start to break off the tails.

Open 8 x 5ml luer-lock syringes

Of 8 syringes, take out the syringe pump of 4 syringes. Place 4 syringe pumps on 100mm or 150mm petri dish

Lock one intact syringe and one syringe with the pump removed to a sterile 22G micro-emulsifying needle on each side

Aliquot cercariae equally into the connected needles. Each syringe should hold ~3ml of cercariae

Place syringe pumps back into the filled syringes and push the pump all the way. The cercariae should pass through the needle to the opposite syringe * Check for any leakage from the locks. If there is a leak, try tightening the lock or do not use that needle

Unlock the empty syringe from the connected needle, place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution

Push the cercariae through the needle, into the tube. Do this for all four needles with max ~5ml per percoll tube

Push the syringes back and forth 12-13 more times

Remove the empty syringe from the connected needle and place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution

Pass the cerariae suspension thought a 22G blunt-end needle in a luer lock syringe as follows:

  If 5ml solution – 15 times using 10ml luer lock syringe

  If 10 ml solution – 30 times using 20ml luer lock syringe

Take a drop of cercariae in a petri dish to check under the microscope if the passages have separated the tails. If there are still many cercariae (more than ~20%), continue with ~5 more passages

Place now-headless cercariae carefully on top of Percoll solution tube using a plastic pasteur pipette (5 ml per Percoll solution tube) or directly into the tube of Percoll solution from the syringe

Centrifuge at 350x g,4°C

Remove from centrifuge carefully. Discard supernatant and the white interface of cercaria tails

Take schistosomula “pellets” and pool them together in a 15ml falcon tube. Top to 15ml with somule wash

Centrifuge at 500x g,4°C

Discard supernatant and add 15ml somule wash

Repeat washing steps 3 times in total

After the last wash, remove as much supernatant as possible and add 6ml of somule media

Dispense 2ml of somules into each well of a 6-well plate and add 4ml somule media to each

Incubate at 37°C in 5% CO₂ incubator

The following day observe the somules to look for contamination, and replace media daily