Subject(s)
Antigen-Presenting Cells/immunology , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Major Histocompatibility Complex , Animals , Antigens, Surface/analysis , Antigens, Surface/immunology , Biotinylation , Cell Membrane/immunology , Cells, Cultured , Culture Techniques/methods , Endocytosis , Enzyme-Linked Immunosorbent Assay/methods , Histocompatibility Antigens Class I/analysis , Histocompatibility Antigens Class II/analysis , Spectrophotometry/methodsABSTRACT
Cell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.
Subject(s)
Biotinylation/methods , Cell Extracts/analysis , Cell Membrane/metabolism , Endocytosis , Membrane Proteins/analysis , Biotin/chemistry , Biotin/metabolism , Cell Extracts/chemistry , Cell Membrane/chemistry , Clathrin/metabolism , Endosomes/chemistry , Endosomes/metabolism , Major Histocompatibility Complex , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Receptors, Transferrin/metabolism , Transferrin/metabolismABSTRACT
Cell surface receptors and antigens, such as TfR and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TfR and MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane proteins.
