H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes in a clear-cut dose dependent manner.

<p>(A) Shown are representative TIRFM images of FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain construct. The FITC-conjugated mouse IgG anti-chicken IgM surrogate molecules were pre-incubated at different concentration (100, 50, 25 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mFI of FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative TIRFM images of IgG surrogate antigen microclusters within the contact interface of chicken DT40 B cell with the PLB membranes tethering FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens. Bar is 1.5 µm. (D) Statistical quantification for the mFI of surrogate antigen microclusters within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.