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 resolution^1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells^{5, 6}, angiogenesis³ and immune cells response^7-9. 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:

1. Grow a cell line expressing Dendra2 protein (as a cytoplasmic marker) to 40 - 80% confluency.
2. Rinse dishes at least 3 times with PBS w/o Ca²⁺ or Mg²⁺.
3. 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.
4. Rinse all the cells off the dish, and use a scraper (rubber policeman) to collect matrix as well.  Add the 5 ml of PBS. Take an aliquot to count during centrifugation.
5. Centrifuge at 800 g for 5 min....

Materials

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
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