Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Summary

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Abstract

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an appropriate immune response to a variety of immunogens^1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be administered intranasally^1,3,4, intratracheally⁵ or as aerosols⁶. Delivery by each route involves distinct technical skills and limitations that need to be considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)⁷, potentially complicating interpretation of the results. In addition, swallowing, sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

Protocol

1. Before the process, prepare and gather the following items

1. Please refer to the image in Figure 1a) and 1b) for building a wooden platform to restrain the mouse during the procedure.
2. Digestion mix for lymph nodes - HBSS + 1.25mg/ml Collagenase TypeIV or 2.5 mg/ml Collagenase D
3. Digestion mix for lungs - HBSS + 1 mg/ml Collagenase
4. Dilute latex beads (1:20) in PBS for intraperitoneal injection to visualize the LDLNs

Note: LDLNs are small and hard to find. To learn how to find them, inject 200μl of the 1:20 diluted red fluorescent latex beads i.p into the mouse. The per....

Discussion

We have utilized this protocol to study trafficking of Bacillus anthracis spores from the lungs to the LDLNs. For similar applications, the number of particles delivered to the lungs should be carefully chosen so that the injected material can be detected in the LDLNs by flow cytometry. We have also successfully used this method for adoptive transfer of cells and labeling of specific lung cell populations using fluorescently labeled antibodies. In addition, this method is routinely used to administer bleomyci.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70μm Cell stariner	BD Biosciences
Aniline Blue	Fisher Scientific	A967-25
Animal Feeding needle	Popper & Sons, Inc.	7920
Collagenase	Sigma-Aldrich	C2139
Collagenase TypeIV	Worthington Biochemical
Collagenase D	Roche Group	11088974103
DPBS	Invitrogen	14190
Fluoresbrite YG Microspheres (0.5μm)	Polysciences, Inc.	17152
HBSS without Calcium chloride and magnesium chloride	Invitrogen	14170
Ketamine HCl (100mg/ml)	Hospira Inc.
Laryngoscope Blade	PennCentury, Inc	For Model LS-1	Refer to www.penncentury.com
Lightweight Fiber Optic Laryngoscope	Welch Allyn	80814
Red Fluorescent Beads (0.5μm)	Invitrogen	F8812	For i.p injection
Xylazine (100mg/ml)	Lloyd, Inc.