TITLE: Schistosoma mansoni cercariae transformation (with needle)
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
Free-living aquatic S. mansoni cercariae transform into the first intramammalian stage,called schistosomula or somules, by burrowing in the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature.  

Transformation of cercariae to schistosomula can be mimicked in the laboratory by triturating cercariae to remove tails, eliminating tails in a percoll gradient, and then culturing the somules in somule media. This method is used when the number of cercariae is high as the percoll gradient results in loss of cercariae. 

Somules can be cultured for several weeks with regular media changes.

[BEFORE_START]
Place 1 sterile 15ml falcon tube on ice per snail to be shed
Pre-chill benchtop centrifuge to 4°C
Prepare 1x DPBS+2% anti-anti and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Prepare somule media and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Prepare somule wash and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)

[GUIDELINES]
Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques

[STEPS]
SECTION: Cercariae collection
1. 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
SECTION: Cercariae collection
2. Using a sterile transfer pipette, dispense cercariae into an autoclaved 50ml beaker
SECTION: Cercariae collection
3. Fill the beaker containing cercariae to 45-50ml with diH2O and incubate for 30 minon ice

5. 
 
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)
SECTION: Cercariae collection
4. Carefully transfer the cercariae to a 50ml Falcon tube and centrifuge at 500 x g, 20 min, 4 °C
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
11. Open 8 x 5ml luer-lock syringes
6. Add 45ml 1x PBS + 2% Anti-Anti and centrifuge at 500 x g, 10 min, 4 °C
7. Repeat this steps 6-7 once more with 1x PBS + 2% Anti-Anti and then twice with somule wash
8. 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)
9. Following centrifugation of the cercariae, remove the supernatant and add 5ml of somule wash per percoll gradient you will use
10. Vortex for 1 min at max speed. This will start to break off the tails.
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
12. Of 8 syringes, take out the syringe pump of 4 syringes. Place 4 syringe pumps on 100mm or 150mm petri dish
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
13. Lock one intact syringe and one syringe with the pump removed to a sterile 22G micro-emulsifying needle on each side
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
14. Aliquot cercariae equally into the connected needles. Each syringe should hold ~3ml of cercariae
SECTION: Alternative tail removal: 20G needle trituration
20. 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
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
15. 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 
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
16. Unlock the empty syringe from the connected needle, place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution 

SECTION: Cercariae tail removal with micro-emulsifying needle trituration
17. Push the cercariae through the needle, into the tube. Do this for all four needles with max ~5ml per percoll tube

SECTION: Alternative tail removal: 20G needle trituration
21. 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
SECTION: Percoll gradient
22. 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
SECTION: Percoll gradient
24. Remove from centrifuge carefully. Discard supernatant and the white interface of cercaria tails
SECTION: Percoll gradient
23. Centrifuge at  350 x g, 5 min, 4 °C
SECTION: Washing somules
25. Take schistosomula “pellets” and pool them together in a 15ml falcon tube. Top to 15ml with somule wash
SECTION: Washing somules
26. Centrifuge at  500 x g, 5 min, 4 °C
SECTION: Washing somules
27. Discard supernatant and add 15ml somule wash
SECTION: Washing somules
28. Repeat washing steps 3 times in total
SECTION: Washing somules
29. After the last wash, remove as much supernatant as possible and add 6ml of somule media
SECTION: Somules in culture
30. Dispense 2ml of somules into each well of a 6-well plate and add 4ml somule media to each
SECTION: Somules in culture
31. Incubate at 37°C in 5% CO2 incubator
SECTION: Somules in culture
32. The following day observe the somules to look for contamination, and replace media daily
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
18. Push the syringes back and forth 12-13 more times
SECTION: Cercariae tail removal with micro-emulsifying needle trituration
19. Remove the empty syringe from the connected needle and place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution