MHC II–EGFP Knock‐in Mouse Model

Abstract The MHC II–EGFP knock‐in mouse model enables us to visualize and track MHC‐II‐expressing cells in vivo by expressing enhanced green fluorescent protein (EGFP) fused to the MHC class II molecule under the MHC II beta chain promoter. Using this model, we can easily identify MHC‐II‐expressing cells, including dendritic cells, B cells, macrophages, and ILC3s, which play a key role as antigen‐presenting cells (APCs) for CD4 + T cells. In addition, we can also precisely identify and analyze APC‐containing tissues and organs. Even after fixation, EGFP retains its fluorescence, so this model is suitable for immunofluorescence studies, facilitating an unbiased characterization of the histological context, especially with techniques such as light‐sheet fluorescence microscopy. Furthermore, the MHC II–EGFP knock‐in mouse model is valuable for studying the molecular mechanisms of MHC II gene regulation and expression by making it possible to correlate MHC II expression (MHC II–EGFP) with surface fraction through antibody detection, thereby shedding light on the intricate regulation of MHC II expression. Overall, this model is an essential asset for quantitative and systems immunological research, providing insights into immune cell dynamics and localization, with a tool for precise cell identification and with the ability to study MHC II gene regulation, thus furthering the understanding of immune responses and underlying mechanisms © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. This article was corrected on 14 April 2024. See the end of the full text for details. Basic Protocol 1 : Characterization of antigen‐specific MHC II loading compartment tubulation toward the immunological synapse Basic Protocol 2 : Characterization of overall versus surface MHC II expression Basic Protocol 3 : Identification and preparation of the lymphoid organs Basic Protocol 4 : Quantification of APC content in lymphoid organs by fluorescence stereomicroscopy Basic Protocol 5 : Quantification and measurement of intestinal lymphoid tissue by light‐sheet fluorescence stereomicroscopy Basic Protocol 6 : Visualization of corneal APCs Basic Protocol 7 : Quantification of MHC II + cells in maternal milk by flow cytometry Support Protocol 1 : Cell surface staining and flow cytometry analysis of spleen mononuclear cells