T cell Receptor Imaging and Activation

The reagents used in our experiments included: OVA_(323–339) (Sigma-Aldrich, St Louis, MO, USA), thapsigargin (TG, stock 1 mM in DMSO, Molecular Probes, Invitrogen, USA), cytochalasin D and nocodazole (Calbiochem, Merck KGaA, Germany). Recombinant murine interferon gamma (IFN-γ) was purchased from Peprotech (Rocky Hill, NJ, USA). For live cell imaging, H57-597-Fab-TCRαβ-Alexa Fluor 647 was purchased from Invitrogen to label TCR as a non-blocking antibody [22 (link)]. For calcium imaging, Calcium Crimson™ was purchased from Invitrogen. The following antibodies (Abs) were used for immunofluorescence: Texas Red-X phalloidin for anti-F-actin, MitoTracker® Green FM (Molecular Probes, Invitrogen, USA), anti-PLCγ1 (sc81, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-PKC-θ (sc212, Santa Cruz Biotechnology), anti-ZAP-70 (Y319, Abcam, Cambridge, MA, USA), anti-ORAI1 (ab59330, Abcam, Cambridge, MA, USA) and anti-PMCA (5 F10, Abcam, Cambridge, MA, USA), Dylight 405-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), Alexa Fluor 555-conjugated goat anti-rabbit IgG (Invitrogen). The following Abs were used for flow cytometry: anti-MHC-II (I-Ab)-FITC, antiCD80-PE, anti-CD86-APC and anti-CCR7-APC (all from eBioscience).