Mouse Spleen Follicular Dendritic Cell Analysis by Flow Cytometry: A Comprehensive Guide

To analyze mouse spleen follicular dendritic cells (FDCs) by flow cytometry, you can follow these steps:\n\n1. Sample preparation: Sacrifice and euthanize the mice according to ethical guidelines and obtain the spleen. Isolate the spleen by dissection and place it in ice-cold phosphate-buffered saline (PBS) to prevent cell activation and clumping.\n\n2. Tissue dissociation: Prepare a single-cell suspension by mechanically dissociating the spleen tissue using frosted glass slides or a gentleMACS Dissociator. Transfer the cell suspension to a sterile tube and centrifuge at low speed (e.g., 300-400 g) for 5 minutes at 4⁴C.\n\n3. Red blood cell (RBC) lysis: Resuspend the cell pellet in RBC lysis buffer to remove the red blood cells. Incubate for the recommended time (usually 5-10 minutes) at room temperature, then add an equal volume of PBS or culture medium to stop the lysis reaction. Centrifuge the cells and resuspend them in PBS.\n\n4. Antibody staining: Prepare a cocktail of fluorescently-labeled antibodies specific for FDC markers. Commonly used FDC markers include CD45, CD21, CD35, and FDC-M1. Incubate the cell suspension with the antibody cocktail for the recommended time at 4⁴C in the dark. Include appropriate isotype controls to account for non-specific binding.\n\n5. Washing and cell fixation: Wash the labeled cells with PBS or staining buffer to remove unbound antibodies. Centrifuge the cells and resuspend them in a fixative solution (e.g., 1-2% paraformaldehyde) to fix the cells and preserve the fluorescence signal. Incubate for the recommended time at room temperature.\n\n6. Flow cytometry analysis: Analyze the fixed cells on a flow cytometer equipped with appropriate lasers and filters for the detection of the fluorochromes used. Set up compensation controls and acquire data using appropriate gating strategies to identify FDCs within the lymphocyte population. Analyze the data using specialized flow cytometry software.\n\n7. Data interpretation: Analyze the flow cytometry data to determine the frequency and phenotype of FDCs in the mouse spleen. Compare the expression levels of FDC-specific markers between experimental groups or different regions within the spleen. Use appropriate statistical tests to assess the significance of any observed differences.\n\nRemember to follow good laboratory practices, maintain sterile conditions, and handle hazardous materials safely throughout the experimental procedure.