Dendra2 Photoswitching through the Mammary Imaging Window
Summary
Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.
Abstract
In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.
Protocol
1. Generation of fluorescent tumors using injection of tumor cells into the mammary fat pad: Grow a cell line expressing Dendra2 protein (as a cytoplasmic marker) to 40 – 80% confluency. Rinse dishes at least 3 times with PBS w/o Ca2+ or Mg2+. Add 3 ml of trypsin per 10 cm dish and incubate at 37 °C until most of the cells detach and then hit the dish against a flat surface to shake it. Rinse all the cells off the dish, and use a scraper (rubber policem…
Acknowledgements
This work was supported by US Department of Defense (BC075554 to B.G. and BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; CA77522 to J.E.S., U54CA126511 to J.C. and B.G.). We thank D. Entenberg for help with microscopy, M. Rottenkolber for help in fabrication of the imaging box and J. Pollard (Albert Einstein College of Medicine) for the F4/80 antibody.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| PBS w/o Ca, Mg | Gibco | 14190-144 | ||
| Trypsin | Gibco | 25300 | ||
| HBSS | Gibco | 14025 | ||
| DMEM media (MDA-MB-231 cells) | Gibco | 11965 | ||
| Isoflurane(Aerrane) | Baxter | # NDC 10019-773-40 | ||
| SCID mouse | NCI | N/A | ||
| Culture dishes | BD Falcon | 353003 | Custom Order | |
| Circular coverslip 8 mm | Scientific, Inc. | 8CIR-1-FIS |
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Tags
Intravital MicroscopyBreast TumorsMiceRatsSingle-cell ResolutionMetastatic BehaviorMigrationInvasionIntravasationAngiogenesisImmune Cells ResponseMammary CarcinomasMammary Imaging Window (MIW)Imaging ParametersDendra2 PhotoswitchingIn Vivo ImagingProtocolManufacturing MIWTumor Cells ImagingCustom Built Imaging BoxMetastatic Behavior
Citer Cet Article
Gligorijevic, B., Kedrin, D., Segall, J. E., Condeelis, J., van Rheenen, J. Dendra2 Photoswitching through the Mammary Imaging Window. J. Vis. Exp. (28), e1278, doi:10.3791/1278 (2009).