Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Solution structure and synaptic analyses reveal determinants of bispecific T cell engager potency.
doi: 10.1073/pnas.2425781122
Figure Lengend Snippet: Fig. 4. TcE-mediated immune synapse formation dynamics and signaling. (A) Schematic overview of T cell–SLB IS experimental system. (B) Representative TIRF images of monofocal and kinapse-like TcE-mediated IS formation at 50 pM TcE on SLBs at 400 (Her2, green) and 200 (ICAM1, yellow) molecules/µm2 after 20 min of interaction, stained for F-actin (magenta). (Scale bar, 5 µm.) (C) Analysis of TcE-mediated immune synapse phenotype frequencies after 20 min of interaction. Numbers in boxes represent the percentage of ISs with bulls-eye actin structures. Mean of three biological replicates. (D) Representative TIRF images of 50 pM TcE-mediated IS formation on SLBs at 400 (Her2) and 200 (ICAM1) molecules/µm2 after 20 min of interaction, stained for F-actin (magenta) and pZap70 (orange). (Scale bar, 5 µm.) (E) Normalized pZap70 levels at the 50 pM TcE-mediated IS at 400 (Left) or 30 (Right) molecules/µm2 of Her2 and 200 molecules/µm2 ICAM1. Yellow solid line = median, White dashed line = quartiles. Three biological replicates, Kruskal–Wallis test, *P < 0.05, **P < 0.005, ***P ≤ 0.0007, and ****P < 0.0001.
Article Snippet: Cells were permeabilized with 0.1% Triton- X100 for 2 min, blocked with 1% BSA in PBS for 20 min, and, where required, subsequently stained with fluorescently labeled Phalloidin and anti- human IgG secondary antibodies for 1 h. To stain for pZap70 and pPLCγ1, cells were blocked for 1 h with 5% BSA in PBS and then either stained with rabbit anti- pPLCγ1 (Cell Signaling, #2821, 1/100) or rabbit anti- pZap70 (Cell Signaling, #2704S, 1/100) overnight at 4 °C.
Techniques: Staining
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