A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.
This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes approximately 24 h while the preparation of egg chambers for live imaging requires only 15-20 min.
Prasad
Mohit
M
Department of Biological Chemistry, Center for Cell Dynamics, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Jang
Anna C-C
AC
Starz-Gaiano
Michelle
M
Melani
Mariana
M
Montell
Denise J
DJ
D000818:Animals; D018929:Cell Culture Techniques; D004331:Drosophila melanogaster / Q000166:cytology* / Q000254:growth & development; D005260:Female; D009865:Oocytes / Q000166:cytology / Q000254:growth & development; D006080:Ovarian Follicle / Q000166:cytology; D010053:Ovary / Q000166:cytology