Alexa fluor 647 mouse anti zap70 py319 syk py352

Jurkat T cells (clone E6-1, ATCC) were cultured in complete RPMI 1640 medium (Life Technologies, France) containing red phenol and L-glutamine supplemented with 1 % glutaMAX (Life Technologies, France) and 10% Fetal Bovine Serum (Life Technologies, France). Cells were in exponential growth phase at the time of experiment. The functionalized glass coverslides formed the bottom of a custom made chamber which was filled with PBS+0,1%BSA buffer. 200 μl of the medium containing cells was added. The cells were allowed to sediment on to the substrate and were incubated for 30 min at 37°C and 5% CO₂. Cells were then fixed by incubation in 2% pre-warmed paraformaldehyde for 30 min at 37 °C, followed by extensive rinsing with PBS. The cells were blocked with 1% BSA overnight and immunostained by incubation with 5 μg/ml of FITC fluorescent Anti-Vβ8 TCR (BD Biosciences, USA) which is directed against the beta chain of the T-cell receptor, or with 20 μg/ml of Alexa Fluor 488 -phalloidin (dissolved in methanol, ThermoFischer, France) which labeled filamentous actin or with Alexa Fluor 647 Mouse Anti-ZAP70 (PY319)/Syk (PY352) (BD biosciences, France) which labeled the kinase ZAP-70 during 30 min. Samples were rinsed extensively before imaging.

Purified anti-mouse CD3ε (clone 145-2C11, #100302) was purchased from BioLegend (San Diego, CA). Alexa Fluor 647 Mouse Anti-ZAP70 (PY319)/Syk (PY352) (#557817) was obtained from BD Biosciences. H-2K^b monomer with the agonist OVA peptide SIINFEKL (OVA-N4) was obtained from the National Institutes of Health (NIH) Tetramer Core Facility (Emory University, Atlanta, GA, USA). The OVA-N4 peptide was synthesized on a Prelude peptide synthesizer (Gyro Protein Technologies) using fluorenylmethyloxycarbonyl chemistry and resuspended in Milli-Q water to 3 mM and sterile-filtered through a 0.22-mm syringe filter.