OMIP‐93: A 41‐color high parameter panel to characterize various co‐inhibitory molecules and their ligands in the lymphoid and myeloid compartment in mice

Abstract

This 41‐color panel has been designed to characterize both the lymphoid and the myeloid compartments in mice. The number of immune cells isolated from organs is often low, whilst an increasing number of factors need to be analyzed to gain a deeper understanding of the complexity of an immune response. With a focus on T cells, their activation and differentiation status, as well as their expression of several co‐inhibitory and effector molecules, this panel also allows the analysis of ligands to these co‐inhibitory molecules on antigen‐presenting cells. This panel enables deep phenotypic characterization of CD4+ and CD8+ T cells, regulatory T cells, γδ T cells, NK T cells, B cells, NK cells, monocytes, macrophages, dendritic cells, and neutrophils. Whilst previous panels have focused on these topics individually, this is the first panel to enable simultaneous analysis of these compartments, thus enabling a comprehensive analysis with a limited number of immune cells/sample size. This panel is designed to analyze and compare the immune response in different mouse models of infectious diseases, but can also be extended to other disease models, for example tumors or autoimmune diseases. Here, we apply this panel to C57BL/6 mice infected with Plasmodium berghei ANKA, a mouse model of cerebral malaria.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon

“…Motivated by an interest for obtaining global snapshots of both innate and adaptative immune systems under various physiological contexts in the murine colon, we developed two antibody panels that target lymphoid and myeloid cell lineages separately. Selected markers were chosen based on existing literature 50 , 52 , 56 , 61 – 66 , also considering that we wanted our panels to be eventually useful in the context of inflammatory conditions as well. As detailed in Tables 1 – 2 , these panels contain 26 antibodies in total (13 lymphoid-specific, 11 myeloid-specific, and 2 shared), mostly targeting extracellular markers (only two are targeting intracellular markers).…”

Development of a customizable mouse backbone spectral flow cytometry panel to delineate immune cell populations in normal and tumor tissues

“…Here, we aimed to design and evaluate a versatile backbone panel for spectral flow cytometry, which allows for robust and customizable immune cell analysis across various tissues and immune microenvironments in mice. While there are already many proposed panels for the profiling of murine immune cells using more than 13 markers ( 38 – 41 , 74 , 75 ), the combinatorial nature of these high-parameter panels and all the complex rules that need to be adhered can make it as challenging to modify only a few parameters to adapt to the research question as it is to build an entirely new panel. These panels were designed as a whole, and most changes can have a profound effect on the overall panel resolution.…”

“…These panels were designed as a whole, and most changes can have a profound effect on the overall panel resolution. Further, most of the current panels were designed for a specific polychromatic flow cytometry experiment, and only a few were tested on a single spectral flow cytometry platform ( 38 – 41 ). To our knowledge, ours is the first murine backbone panel validated across different spectral instruments; thus, this panel is a valuable resource for researchers who have access to any of the current spectral flow cytometer systems.…”

“…Since 2022, a few multiparameter panels for spectral cytometers have been introduced to study subtypes of immune cells on murine samples ( 38 – 43 ). Although some of the panels include markers for both myeloid and lymphoid populations, they were optimized for a specific type of organ or for a single instrument ( 41 , 42 ) and there is no agreement on the gating strategy for essential immune subsets ( 41 , 44 ). Furthermore, based on the experience of the authors, high dimensional panels such as these including 20-40 markers are difficult to customize and/or optimize.…”

OMIP‐095: 40‐Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues

“…OMIPs −057, −059, and − 061 explore γδT cell development in the thymus, progenitor composition in the bone marrow, and antigen presenting cell frequency in virally infected lymph nodes respectively; thus, they share small overlaps in the populations and tissues explored in this work [135–137]. Lastly, OMIP‐93 shares similarity in complexity and panel purpose [138].…”