Journal: Frontiers in Immunology

Article Title: High-Affinity Ligands Can Trigger T Cell Receptor Signaling Without CD45 Segregation

doi: 10.3389/fimmu.2018.00713

Figure Lengend Snippet: Calcium flux in Jurkat T cells stimulated with membrane-tethered anti-CD3 scFv. Jurkat T cells were dropped on planar lipid bilayers decorated with anti-CD3 scFv in the presence or absence of Lck inhibitor (PP2). The emission was measured from Fura-2AM labeled Jurkat T cells as the ratio between 340 and 380 nm at different times (min). (A) Ca 2+ flux measured from individual Fura-2AM stained Jurkat T cells. A heat map for Ca 2+ from individual Jurkat T cells over time. Mean ratiometric fluorescence of Fura-2AM loaded Jurkat T cells triggered by membrane-tethered OKT3-1 (B) , OKT3-CD43 (C) , OKT3 MA -1 (D) , OKT3 MA -CD43 (E) , or anti-DNS control scFv (F) ( n = 52 and 30 cells without PP2 and with PP2 inhibition, respectively). Bars, SD.

Article Snippet: ScFv ligand densities on the surface of lipid bilayers were estimated based on the fluorescence intensity of stained lipid bilayer-coated glass beads that were decorated with scFv relative to Fc-specific capture antibody bead standards (Quantum TM Simply Cellular ® , Bangs Laboratories, Inc.) as described by Dustin et al. ( ).

Techniques: Membrane, Labeling, Staining, Fluorescence, Control, Inhibition