Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Summary

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Abstract

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

Introduction

Following inhalation of pro-inflammatory stimuli such as lipopolysaccharide (LPS), there is a coordinated movement of immune cells into, within, and from the lung. For example, neutrophils are rapidly recruited to the lung parenchyma and airway. In addition, some professional antigen presenting cells known as conventional dendritic cells (cDCs) undergo a relatively complex migration pattern^1,2. cDCs can be identified using flow cytometry, based in part on their display of the surface marker, CD11c. Distinct subsets of DCs can be distinguished by the differential surface expression of CD103 and CD11b

Protocol

Animal experimental procedures described in this paper were approved by the NIEHS Animal Care and Use Committee (IACUC).

1. Lung Preparation

1. House mice between 6 and 12 weeks of age in specific pathogen-free conditions in accordance with the guidelines provided by the Institutional Animal Care and Use Committees.
   NOTE: Imaged mice can be either naïve, or treated, depending on each researcher's specific interests. Here, we describe immune cell localization in naïve mice and in mice treated with 100 µg ovalbumin (OVA) and 0.1 µg lipopolysaccharide (LPS), using phosphate buffered saline (PBS) as a veh....

Results

To identify the location of two DC subsets, CD11b^hi cDCs and CD103⁺ cDCs, PCLS from C57BL/6 mice were cut and stained with monoclonal antibodies (mAbs) specific to CD11c, CD88, CD103, and CD324 (E-cadherin). Antibodies to CD324 stain airway epithelial cells, and CD88 is displayed on macrophages and neutrophils, but not cDCs⁸. This allowed us to distinguish cDCs from CD11c⁺ macrophages, and to observe the spatial relationship of each.......

Discussion

The protocol described here was originally developed to visualize the locations of two subsets of cDCs within the lung. However, this protocol can be readily adapted to study many different cell types, while maintaining cell viability and the three-dimensional architecture of the lung. The latter feature is an important advantage over cell culture systems and facilitates identification of rare cell types. The method relies on the generation of PCLS from the lung, and an appropriate combination of antibodies to identify s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J mice	Jackson Laboratory	000664
Prox1-TdTomato transgenic mice	Jackson Laboratory	018128	B6;129S-Tg(Prox1-tdTomato)12Nrud/J
OT-II OVA-specific TCR x Nur77-GFP transgenic mice	Jackson Laboratory	004194, 016617	B6.Cg-Tg(TcraTcrb)425Cbn/J x C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J
Rag1 knock-out mice	Jackson Laboratory	002216	B6.129S7-Rag1tm1Mom/J
Ovalbumin, Low Endo, Purified	Worthington Biochemical Corporation	LS003059
Lipopolysaccharides from Escherichia coli	Sigma-Aldrich Co.	L2630-25MG
Polyethylene tubing (Non-Sterile) 100 ft	BD Diagnostic Systems	427421	0.86 mm inside diameter, 1.27 mm outside diameter
GeneMate Sieve GQA Low Melt Agarose	BioExpress	E-3112-125	2% solution dissolved in PBS at 70 °C and held at 40 °C.
Compresstome VF-300	Precisionary Instruments, Inc.	VF-300
Double Edge Stainless Razor Blade	Electron Microscopy Sciences	72000	Disposable; 250/box. Blade should be changed for every lung.
Krazy Glue All Purpose Instant Gel	VWR	500033-484	Commonly available for $3/tube in local drugstores
Leibovitz's L-15 Medium, no phenol red	ThermoFisher Scientific	21083027
Normal Rat Serum (NRS)	Jackson ImmunoResearch Inc.	012-000-120
Normal Mouse Serum (NMS)	Jackson ImmunoResearch Inc.	015-000-120
Fetal bovine serum (FBS)	Hyclone	SH30071.03HI
Staining Buffer	Made in House	N/A	PBS w/ 0.5% bovine serum albumin, 0.1% NaN3, pH 7.4
Fc Blocker (anti-CD16/32 antibodies)	Made in House	N/A	Supernatant of cultured hybridoma cell line 2.4G2
Anti-mouse CD11b eFluor 450	eBioscience	48-0112-80	Anti-mouse CD11b eFluor 450 (clone: M1/70)
Anti-mouse CD11c Brilliant Violet 605	BioLegend	101237	Brilliant Violet 605 anti-mouse CD11c (clone: M1/70)
Anti-mouse CD11c Phycoerythrin	eBioscience	12-0114-82	PE conjugated anti-mouse CD11c (clone: N418)
Anti-mouse CD11c Allophycocyanin	BD Phamingen	550261	APC-labeled anti-mouse CD11c 9clone: HL3)
Anti-mouse CD88 Phycoerythrin	BioLegend	135806	PE anti-mouse CD88 (clone: 20/70)
Anti-mouse CD103 Allophycocyanin	eBioscience	17-1031-82	Anti-mouse CD103 APC (clone: 2E7)
Anti-mouse CD90.2/Thy1.2 eF450	eBioscience	48-0902-82	Anti-mouse CD90.2 eFluor 450 (clone: 53-2.1)
Anti-mouse CD172a/Sirp1a Allophycocyanin	eBioscience	17-1721-82	Anti-mouse CD172a APC (clone: P84)
Anti-mouse CD324 Brilliant Violet 421	BD Horizon	564188	BV421 mouse anti-E Cadherin (clone: 5E8 also known as 5E8-G9-B4)
Anti-mouse CD324 Alexa Fluor 488	eBioscience	53-3249-82	Anti-CD324 (E-Cadherin) Alexa Flour 488 (clone: DECMA-1)
Anti-mouse CD324 Alexa Fluor 647	eBioscience	51-3249-82	Anti-CD324 (E-Cadherin) Alexa Flour 647 (clone: DECMA-1)
Glass Bottom Microwell Dishes 35 mm petri dish, 14 mm Microwell, No. 1.5 coverglass	MatTek Corperation	P35G-1.5-14-C
Nunc Lab-Tek Chambered Coverglass	ThermoFisher Scientific	155411PK	Pack of 16
15 mm Coverslip, No. 1.5 Glass Thickness	MatTek Corperation	PCS-1.5-15
Bare Platinum Wire	World Precision Instruments	PTP201	0.020" (0.5 mm) diameter cut into ~1 cm long pieces and bent into an "L" shape
ProLong Gold Antifade Mountant	ThermoFisher Scientific	P36934	Keep at 4 °C, warm to room tempterature before use.
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, *LDEV-Free	Corning	356231
Zeiss 880 multi-photon laser-scanning microscope	Carl Zeiss		Zen Black software version 8.1, 2012 (Zeiss)
Plan-Apochromat 20X/0.8 M27 objective lends	Carl Zeiss	420650-9901-000