Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Summary

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Abstract

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Introduction

Osteoarthritis (OA) is a debilitating and painful disease involving various pathologies of all tissues associated with the joint¹. Affecting approximately 3.8% of the global population², OA is one of the most prevalent musculoskeletal diseases and it is to become the 4^th leading cause of disability worldwide by 2020³. Post-traumatic OA occurs after a joint injury and accounts for at least 12% of all OA and up to 25% of OA in susceptible joints such as the knee^4,5. Furthermore, joint injury increases the lifetime risk of OA by mo....

Protocol

Northern Sydney Local Health District Animal Ethics Committee has approved all procedures mentioned in this protocol. Mice are housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Health and Medical Research Council of Australia Revised 2010). For all experiments 10-12-week-old, male C57BL/6 mice were utilized.

NOTE: To induce post-traumatic OA, surgical destabilization of the medial meniscus (DMM) in the right stifle joint was performed. Detailed information regarding this animal model was published by Glasson et al.¹⁸. In short, general anesthesia i....

Results

Representative results from both the monocyte subset panel and T-cell subset panel are described below.

Figure 1 illustrates the hierarchical gating strategy for the monocyte subset panel on immune cells gathered from bone marrow of DMM treated animals. The same strategy was used and verified in all other tissue types. When setting up the experiment, the Forward Scatter Area (FSC-A) and Side Scatter Area (SSC-A) voltage was determined for each tissue type to ident.......

Discussion

The methods described in this protocol have been designed and tested to reliably identify various subsets from both monocytes/macrophages and T-cells within the murine spleen, bone marrow, lymph nodes, and synovial tissue in a murine model of osteoarthritis (OA). The current protocol can easily be modified to investigate different tissue types, or other cell types by exchanging antibodies, and can be used for alternative murine models of OA. When testing other tissue types, it is critical to test the specificity of each .......

Materials

Name	Company	Catalog Number	Comments
APC anti-mouse CD194 (CCR4)	BioLegend	131212	T-Cell Panel
Brilliant Stain Buffer Plus 1000Tst	BD	566385	Buffers
Fixable Viability Stain 510, 100 µg	BD	564406	T-Cell Panel
Fixable Viability Stain 510, 100 µg	BD	564406	Monocyte Panel
Liberase, Research Grade	Roche	5401127001	Enzyme for synovial tissue
Ms CD11b APC-R700 M1/70, 100 µg	BD	564985	Monocyte Panel
Ms CD11C PE-CF594 HL3, 100 µg	BD	562454	Monocyte Panel
Ms CD183 BB700 CXCR3-173, 50 µg	BD	742274	T-Cell Panel
Ms CD206 Alexa 647 MR5D3, 25 µg	BD	565250	Monocyte Panel
Ms CD25 BV605 PC61, 50 µg	BD	563061	T-Cell Panel
Ms CD3e APC-Cy7 145-2C11, 100 µg	BD	557596	T-Cell Panel
Ms CD4 PE-Cy7 RM4-5, 100 µg	BD	552775	T-Cell Panel
Ms CD44 APC-R700 IM7, 50 µg	BD	565480	T-Cell Panel
Ms CD62L BB515 MEL-14, 100 µg	BD	565261	T-Cell Panel
Ms CD69 BV711 H1.2F3, 50 µg	BD	740664	T-Cell Panel
Ms CD80 BV650 16-10A1, 50 µg	BD	563687	Monocyte Panel
Ms CD8a BV786 53-6.7, 50 µg	BD	563332	T-Cell Panel
Ms F4/80 BV421 T45-2342, 50 µg	BD	565411	Monocyte Panel
Ms Foxp3 PE MF23, 100 µg	BD	560408	T-Cell Panel
Ms I-A I-E BV711 M5/114.15.2, 50 µg	BD	563414	Monocyte Panel
Ms Ly-6C PE-Cy7 AL-21, 50 µg	BD	560593	Monocyte Panel
Ms Ly-6G APC-Cy7 1A8, 50 µg	BD	560600	Monocyte Panel
Ms NK1.1 BV650 PK136, 50 µg	BD	564143	T-Cell Panel
Ms ROR Gamma T BV421 Q31-378, 50 µg	BD	562894	T-Cell Panel
Red Blood Cell Lysing Buffer	N/A	N/A	Buffers	Description in: Immune Cell Isolation from Mouse Femur Bone Marrow / Xiaoyu Liu and Ning Quan/ Bio Protoc. 2015 October 20; 5(20): .
Transcription Factor Buffer Set 100Tst	BD	562574	Buffers