Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging
Published: April 30, 2023
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. Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic …
Representative 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) 20 G needle inserted 4-5 mm into the trachea. (D) Instillation of 400 µl 2% low-melting-tem…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging. J. Vis. Exp. (Pending Publication), e20232, doi: (2023).