Goat anti rabbit apc

Trout splenocytes were isolated and seeded in complete MGFL-15 medium (MGFL-15 supplemented with P/S, 100 μg/ml gentamicin, 10% newborn calf serum (Gibco) and 5% carp serum). Cells were incubated for 24 h with complete media containing IL-6, LPS or control media alone. Following stimulation, cells were fixed in 1% formaldehyde, and washed twice in PBS with 2% calf serum and 0.1% saponin (permeabilization buffer). To determine nuclear translocation in IgM⁺ cells, splenocytes were stained with anti-p65 (Santa Cruz Biotechnology) and anti-trout IgM (1.14) for 30 min at 4 °C followed by 20 min at RT. Following the primary staining, cells were washed and stained with goat anti-rabbit APC (Jackson ImmunoResearch) and rabbit anti-mouse FITC (Jackson ImmunoResearch). Prior to acquisition, Hoechst33342 nuclear stain (Molecular Probes) was added as per the manufacturer’s recommendations. Data were collected on an ImageStream MKII and analyzed using IDEAS software (Amnis), as described previously11 (link).

HEK-293T cells (human embryonic kidney 293T cells) were cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% (v/v) FBS (fetal bovine serum). JE6 and P116 cells were cultured in RPMI 1640 (Roswell Park Memorial Institute) with 10% (v/v) FBS in the presence or absence of inhibitors. Anti-Myc (9E10), anti-Zap70 (1E7.2), and ProteinG Plus agarose were purchased from SantaCruz Biotechnology. Anti-FLAG and anti-phospho 44/42 MAPK were obtained from Cell Signaling Technology and biotinylated anti-CD3ε (UCHT1) from BioLegend. Anti-β-Actin antibody, MG132, N-acetylcysteine and Geldanamycin were purchased from SigmaAldrich and Cycloheximide from Carl Roth. DCP-Bio1 was from MerckMillipore, Streptavidin-HRP, was from ThermoFischer, [S35]Methionine/Cysteine was from Hartmann Analytic GmbH and the goat-anti rabbit APC was from Jackson ImmunoResearch.