Haemonchus contortus L3 ex-sheathing.

Glucose Tyrode's recipe

HcL3 media recipe

Materials

• 2% bleach solution
• Glucose Tyrode's buffer
• HcL3 media
• 50 mL conical tubes

Protocol

1. Remove Glucose Tyrode's buffer and HcL3 media from the 4°C fridge and aliquot into 50 mL conical tubes. Place the aliquots in a 37°C incubator 1 hour before starting the ex-sheathing process.
2. Remove the Haemonchus from the 4°C deli fridge 1 hour before starting, draw off the volume needed for your assay, and add it to a 50 mL conical tube.
3. Calculate the volume based on the concentration of L3s you will need for your assay.
4. Add DI water up to the 45 mL line, then add 5 mL of the 2% bleach solution and mix well.
   • You can use a 15 mL tube or a 1.5 mL tube instead, but keep the bleach solution in a 1:9 ratio.
5. After incubating for 5 minutes, spin the tube at 1,000 rpm for 3 minutes.
6. Draw off the solution without disturbing the HcL3 pellet and wash with Glucose Tyrode's.
7. Repeat steps 4 and 5 three times.
8. After the last spin, draw off as much of the Glucose Tyrode's as possible, then add HcL3 media up to the volume needed for your assay.