A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice
Transcript
Take an anesthetized mouse. Inject Mycobacterium bovis BCG suspension into the hind footpad.
Skin dendritic cells recognize and phagocytose the mycobacteria.
Further, the processed mycobacterial components are presented on MHC class-II molecules.
Inject the cell-permeable CFSE dye into the same footpad.
Intracellular esterases cleave CFSE into a cell-impermeable fluorescent product, staining the cells.
Further, activated dendritic cells migrate via lymphatic vessels to the popliteal lymph node, activating T cells.
Surgically excise the popliteal lymph node and homogenize it through a strainer, obtaining a single-cell suspension.
Centrifuge and resuspend the cells in a buffer.
Transfer cells to a flow cytometry tube. Centrifuge and treat the cells with an antibody cocktail.
Fc-blocking antibodies block Fc receptors, while fluorophore-tagged antibodies bind to surface markers CD11c and MHC class-II molecules on immune cells.
Using flow cytometry, identify the CFSE-labeled skin dendritic cells with high MHC-II and low CD11c expression, confirming their migration to the lymph node.
