Schistosoma mansoni cercariae transformation (without needle)
Sarah K Buddenborg
Abstract
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 centrifuging cercariae to remove tails and then culturing the somules in somule media. This method is particularly useful when the number of cercariae is low (i.e. clonal cercariae from an individual snail).
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)
Steps
Cercariae collection
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 sterile 15ml falcon tubes on ice
After collecting cercariae, adjust the volume in each 15ml falcon tube to 15ml with sterile water
Incubate tubes containing cercariae for 0h 30m 0s``On ice
Cercariae tail removal
Centrifuge the cercariae 2200rpm,4°C
Quickly remove the supernatant and resuspend the pellet in 10ml pre-warmed 1x PBS + 2% PSF (see "MATERIALS" section for recipes) by gently inverting the tube (do not use pipet to mix)
Centrifuge the cercariae 1500rpm,4°C
Repeat steps 6 and 7 six more times
Somules in culture
Resuspend pellet of somules in ~5ml pre-warmed 37°C somule media (see "MATERIALS" section for recipes)
Place somules in a 6-well plate and top up each well with pre-warmed 37°C somule media so each has a total of approximately 4ml media
Incubate at 37°C, 5% CO20h 30m 0s
Optional: the following day, remove tails with transfer pipet (the tails will have floated to the top of the water column in the wells)
