Abstract
Cultured Drosophila melanogaster S2 and S2R+ cell lines have become important tools for uncovering fundamental aspects of cell biology as well as for gene discovery. Despite their utility, these cell lines are nonmotile and cannot build polarized structures or cell-cell contacts. Here we outline a previously isolated, but uncharacterized, Drosophila cell line named Dm-D17-c3 (or D17). These cells spread and migrate in culture, form cell-cell junctions and are susceptible to RNA interference (RNAi). Using this protocol, we describe how investigators, upon receiving cells from the Bloomington stock center, can culture cells and prepare the necessary reagents to plate and image migrating D17 cells; they can then be used to examine intracellular dynamics or observe loss-of-function RNAi phenotypes using an in vitro scratch or wound healing assay. From first thawing frozen ampules of D17 cells, investigators can expect to begin assaying RNAi phenotypes in D17 cells within roughly 2–3 weeks.
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Acknowledgements
We thank members of the Rogers lab for helpful discussions regarding the application of this protocol. We would also like to acknowledge M. Peifer for the generous gift of Canoe antibody and the DGRC for cell lines and helpful protocols.
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J.D.C. performed the protocol under the supervision of S.L.R. J.D.C. and S.L.R. wrote the protocol manuscript.
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The authors declare no competing financial interests.
Supplementary information
Supplementary Video 1. D17 wound migration.
D17 cells treated with control dsRNA and imaged in intervals of 10 minutes over 16 hours. (MOV 8333 kb)
Supplementary Video 2. D17 single cell migration.
D17 cell transiently transfected with pMT GFP-Orbit and imaged every 3 seconds using spinning disc confocal microscopy. GFP-Orbit is a Drosophila microtubule plus end protein that labels the growing ends of microtubules in cells. (MOV 1660 kb)
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Currie, J., Rogers, S. Using the Drosophila melanogaster D17-c3 cell culture system to study cell motility. Nat Protoc 6, 1632–1641 (2011). https://doi.org/10.1038/nprot.2011.397
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DOI: https://doi.org/10.1038/nprot.2011.397
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