Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

Podsumowanie

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Streszczenie

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

Wprowadzenie

The murine respiratory tract contains a unique immune system responsible for fighting pathogens and maintaining immune homeostasis. The pulmonary immune system consists of cellular populations with significant heterogeneity in their phenotype, function, origin, and location. Resident alveolar macrophages (AMs), originated mainly from fetal monocytes, reside in the alveolar lumen¹, while bone marrow-derived interstitial macrophages (IMs) reside in the lung parenchyma². IMs can be further subclassified by the expression of CD206. CD206⁺ IMs populate the peribronchial and perivascular area, while CD206^-

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Protokół

All studies and experiments described in this protocol were conducted under guidelines according to the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center. Six to ten weeks old C57BL/6 mice of either sex were used to develop this protocol.

1. Surgical excision and tissue preparation

1. Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol; Table of Materials).
   NOTE: CO₂ asphyxiation should be avoided in lung studies as it might cause lung injury and alter the features and properties of lung immun....

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Wyniki

Gating strategy
The first step of our gating strategy is the exclusion of the debris and doublets (Figure 1A). Careful exclusion of doublets is critical to avoid false-positive populations (Supplemental Figure S2). Then, immune cells are identified using CD45⁺, a marker for hematopoietic cells (Figure 1B). The live-dead stain can be added to exclude dead cells. However, this protocol results in the death of.......

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Dyskusje

Identification of pulmonary immune cells can be challenging because of the multiple immune cell types residing in the lung and their unique immunophenotypic characteristics compared to their counterparts residing in other tissues. In several pathologic conditions, cells with distinct phenotypic features appear in the lungs. For example, bleomycin-induced lung injury results in the recruitment of circulating monocyte-derived macrophages in the alveolar space, where they can remain for as long as one year and even persist .......

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Materiały

Name	Company	Catalog Number	Comments
10 mL syringe plunger	EXELINT	26265
18 G needles	BD Precision Glide Needle	305165
21 G needles	BD Precision Glide Needle	305195
50 mL conical tubes	Falcon	3520
70 μm cell strainer	ThermoFisher	22363548
96-well plates	Falcon/corning	3799
ACK Lysing Buffer	ThermoFisher	A10492-01
anti-mouse CD11b	Biolegend	101215	For details see Table 2
anti-mouse CD11c	Biolegend	117339 / 117337	For details see Table 2
anti-mouse CD45	Biolegend	103115	For details see Table 2
anti-mouse CD64	Biolegend	139319	For details see Table 2
anti-mouse CD68	Biolegend	137009	For details see Table 2
anti-mouse GR-1	Biolegend	108433	For details see Table 2
anti-mouse Siglec F	Biolegend	155503	For details see Table 2
AVERTIN	Sigma-Aldrich	240486
B220	Biolegend	103228	For details see Table 2
Bovine Serum Albumin (BSA)	Sigma-Aldrich	9048-46-8
CD103	Biolegend	121405 / 121419	For details see Table 2
CD24	Biolegend	138503	For details see Table 2
CD3	Biolegend	100205	For details see Table 2
Centrifuge
Collagenase Type 1	Worthington Biochemical Corp	LS004196
CX3CR1	Biolegend	149005	For details see Table 2
DNase I	Millipore Sigma	10104159001
Ethanol
F4/80	Biolegend	123133	For details see Table 2
FcBlock (CD16/32)	Biolegend	101301	For details see Table 2
Fetal Bovine Serum	R&D Systems
Fine Serrated Forceps	Roboz Surgical Instrument Co
Foxp3 / Transcription Factor Staining Buffer Set	ThermoFisher	00-5523-00
Futura Safety Scalpel	Merit Medical Systems	SMS210
Live/Dead Fixable Far Read Dead Cell Stain Kit	ThermoFisher	L34973	For details see Table 2
MERTK	Biolegend	151505	For details see Table 2
MHC-II	Biolegend	107621	For details see Table 2
NK1.1	Biolegend	108705	For details see Table 2
Orbital Shaker	VWR	Model 200
Petri dish	Falcon	351029
Refrigerated benchtop centrifuge	SORVAL ST 16R
Small curved scissor	Roboz Surgical Instrument Co