Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging

Abstract

Source: Van den Bijgaart R.J.E. et al., Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment, J. Vis. Exp. (2016)

This video describes a method of preparing lungs for ex vivo live imaging to study interactions between cancer cells and stromal cells, using agarose for inflation. In the sample protocol, agarose inflation procedure is described before observing live cellular interactions.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Lungs for Ex vivo Live Imaging

NOTE: Try to work as sterile and careful as possible to avoid unnecessary challenges of the immune cells within the lungs.

1. Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic permitted by the animal protocol approved by IACUC, e.g., 1 ml of 2.5% Avertin. Wait for the mouse to stop breathing and be completely non-responsive to ....

Results

Figure 1. Protocol for preparation of lungs for live imaging. (A) Exposure of the trachea after preparation of the mouse. (B) Small snip made in the exposed trachea parallel to the cartilaginous rings. (C

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthetic	NA	NA	Anesthesia approved by IACUC, used for anesthesia and/or euthanesia
PBS, USP sterile 	Amresco INC 	K813-500ML 	Ultra pure grade for i.v. injection
1X PBS 	UCSF cell culture facility	NA
Styrofoam platform	NA	NA	Will be used as dissection board
Fine scissors sharp  	Fine Science Tools	14060-11
20 G x 1 needle, short bevel 	BD 	305178
Forceps  	Roboz Surgical Store	RS-5135
1 mL syringe without needle 	BD 	309659
Low-melting-temperature agarose 	Lonza 	50111	To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 °C water bath until used for inflation.
RPMI-1640 medium without phenol red 	Life Technologies 	11835-030
24 well Imaging plate 	E&K scientific	 EK-42892
Glass cover slides, 15 mm 	Fisher Scientific 	22-031-144
Digital CO2 and temperature controller 	Okolab 	DGTCO2BX 	http://www.oko-lab.com
Climate chamber 	Okolab 	NA	http://www.oko-lab.com
Cell Observer spinning disk confocal microscope	 Zeiss	NA
Air 	UCSF 	NA
Oxygen	UCSF 	NA
Carbon dioxide	UCSF 	NA