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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.
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.
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 4th 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 knee4,5. Furthermore, joint injury increases the lifetime risk of OA by more than five times6. Not all injuries with apparently similar instability will go on to develop OA, and therefore defining factors that drive the long-term OA-risk remains challenging. It is crucial in order to develop effective treatments to prevent and/or treat post-traumatic OA, to investigate and better define the injury-specific pathology, causes, and mechanisms that predispose to OA1.
OA and its defining cartilage destruction was previously attributed entirely to mechanical stress and, thus, OA was considered a non-inflammatory disease2. However, more recent studies have shown an inflammatory infiltration of synovial membranes and an increase of inflammatory cells in the synovial tissue in patients with OA compared to healthy controls2, shedding light on an inflammatory component as a potential driving force in OA. Further studies indicated that abnormalities in both the CD4+ and CD8+ T-cell profile as well as monocytes/macrophages of the innate immune system may contribute to the pathogenesis of OA2,7. Detailed investigations into these abnormalities revealed relevant roles for various T cell subsets2, such as Th18, Th29, Th178 and T regulatory (Treg) populations10,11. Despite this compelling evidence, the causal relationship between the alteration of T-cell responses and the development and progression of OA is still unknown2.
In addition to specific T-cells having a role in OA, recent studies suggest that differentially polarized/activated macrophages may be associated with pathogenesis of OA12. In particular, macrophages originating from blood monocytes accumulate in the synovium and polarize into either classically activated macrophages (M1) or alternatively activated macrophages (M2) during OA development, implying a correlation between monocyte derived macrophages and OA13. In contrast, certain subsets of macrophages populate organs early during development and self-sustain their numbers in a monocyte independent matter14. Recently, a joint protective function mediated by a tight-junction barrier was shown for these synovial-tissue-resident macrophages (STRMs)14. These findings indicate that abnormalities in particular macrophage subsets may play a crucial role during development of OA. However, the interactions between this inflammatory cellular response and the structural changes in the joint subsequent to trauma is unknown.
Historically, analysis of immune cells in the synovial tissue was restricted to immunohistochemistry (IHC) or mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR) approaches15,16. However, both IHC and RT-PCR lack the ability to identify multiple different cell types and their subsets simultaneously, thus, limiting the applicability of these methods. Furthermore, IHC is limited to analysis of small samples of tissue and may miss focal inflammatory cell accumulations. Over the last several years, a myriad of surface markers for various cell types have been developed, and subsets of immune cells can now be reliably identified by distinct combinations of these markers. Due to steady technical progress, flow cytometers are now capable of identifying a multitude of different fluorochromes simultaneously enabling analysis of large multicolor antibody panels.
Flow cytometry provides investigators with a powerful technique that allows simultaneous identification and quantification of a multitude of immune cells and their subsets at the single cell level. We have developed and optimized both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra/intracellular staining assay to identify T-cells and their subsets within 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 models17.
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 is induced in an induction chamber using isoflurane and thereafter maintained using a nose cone. The surgical leg is shaved with a razor blade and the surgical site is washed and swabbed with ethanol to minimize contamination. The animal is then moved to the operating microscope and placed on a sterile towel and the leg draped with sterile paper drape to isolate the surgical site and minimize contamination. Using the microscope, a 0.5 cm medial para-patella arthrotomy is made, the patella luxated laterally, and the infra-patella fat pad elevated to expose the medial menisco-tibial ligament, which is transected with dissecting forceps. The joint is flushed with sterile saline to remove any blood and the wound is closed in three layers – joint capsule, subcutaneous tissue (using suture material) and skin (using surgical tissue glue). Methods described in this protocol, however, can be applied to other models and methods for inducing OA. OA can be induced in either side of the animal, and when harvesting tissues, it is important to harvest the ipsilateral (draining) lymph nodes.
1. Isolation of the spleen, contralateral bone marrow, ipsilateral lymph nodes draining the stifle and synovial tissue
2. Generation of single cell suspensions from each tissue
NOTE: In order to ensure sufficient cell numbers for flow analysis synovial tissues from two mice need to be pooled. In the current protocol, pool all tissues from the same two mice in order to maintain analogy. Furthermore, iliac and inguinal lymph nodes were combined for each animal resulting in a total of 4 lymph nodes for each sample. In general, cell numbers in spleen, bone marrow and lymph nodes from one animal are sufficient to conduct flow analysis and the protocol can be applied. However, when using tissues from only one animal lysing times might need to be adjusted.
3. Allocation of cells
4. Monocyte Subset Panel
5. T Cell subset panel
6. Compensation, appropriate controls and gating
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...
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 ...
The authors have nothing to disclose.
We would like to thank Andrew Lim, Ph.D. and Giles Best, Ph.D. for their help in setting up the flow cytometer. This project was supported by the Deutsche Forschungsgemeinschaft (DFG) (DFG-HA 8481/1-1) awarded to PH.
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 |
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