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 ? ~1 mm dwells in the soil completely sequenced genome simple body plan

Design Project Goal: design an isolated, in vitro C. elegans gonad preparation protocol.

Purposes for the purpose of characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy This includes: Voltage sensitive dyes, pH sensitive dyes, Ca+ sensitive dyes, etc.

1st 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 and gonad does not completely remove itself without manipulation.

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

2nd 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

Worm Response to Buffer Any of the previous listed buffers would sustain function to a point. The worm gonad needs the the salts to mimic the interstitial fluid of the worm. The gonad must also have an energy source . . .this is where the glucose comes in.

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.

3rd Phase - Imaging optimal dye loading on perfusion chamber DIC image acquisition Imaging with argon laser confocal microscope Tie the process together and formalize protocol