Development of an Isolated in Vitro C elegans

Development of an Isolated, in Vitro C. elegans Gonad Preparation Adam Broslat Advisor: Dr. Kevin Strange Professor of Anesthesiology and Pharmacology

What are C. elegans ?

Design Project Goal: design an isolated, in vitro C. elegans gonad preparation protocol. Purpose: characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy.

1 st Phase – Micro-dissection procedure • The nematode's gonad will be isolated in such a way not to harm the physiology of the gonad. Gonad operates independently of the worm Problems: The intestines “cloud” the view of gonad after dissection.

Solutions to Date • Incisions made with a modified injection needle (red line) • Gonad is half extracted through depressurization • The other half is forced by suction using micropipettes

2 nd Phase – Functional Buffer • The worm and/or gonad must be placed in a buffer solution that promotes normal gonad function Problems: Worms are extremely active in buffer. Buffer allows floating and movement.

Buffer Recipes

Solutions to Date • . 1% Tricaine and. 01% Tetramasole anesthetic was added to chilled buffer for stabilization. • Veterinary glue was used on the glass of perfusion chamber to secure gonad.

3 rd Phase – What's to come • optimal dye loading on perfusion chamber • DIC image acquisition • Imaging with argon laser confocal microscope • Tie the process together and formalize protocol