Our understanding of cell-to-cell interactions has been significantly improved in the past years with the help of TIRFM in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes. In TIRFM experiments, it is a challenge to uniformly present ligand molecules on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of PLB membranes containing nickel salt. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on nickel-containing PLB membranes. We systemically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens show good lateral mobility and are uniformly distributed on PLB membranes. Not IgM molecules, nor Fab or F(ab’)2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust way to tether IgG surrogate antigens or other molecule fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.
- Copyright © 2013 by The American Association of Immunologists, Inc.