Anti ccr7 apc

Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).

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).