Anti p tyr

Conjugates between Jurkat cells and SEE-pulsed Raji B cells were carried out as previously described (Finetti et al., 2009 (link)). Raji cells were pulsed for 2 h with 10 μg/ml SEE (Toxin Technology, Sarasota, FL, United States) and labeled with 10 μM Cell Tracker Blue (Molecular Probes) for the last 20 min. Conjugates between T cells and unpulsed B cells were used as negative controls. SEE-pulsed or unpulsed Raji B cells were mixed with Jurkat T cells (1:1) and conjugates analyzed 15 min after their formation. Samples were allowed to adhere for 15 min on poly-L-lysine (Sigma-Aldrich)-coated wells of diagnostic microscope slides (ThermoFisher Scientific), then fixed by immersion in methanol for 10 min at -20°C. Following fixation, samples were washed in PBS and incubated with anti-pTyr (Cell Signaling, #8954) at 10 μg/mL and anti-CD3ζ at 15 μg/mL (SantaCruz, #sc-1239) in PBS 1X overnight at 4°C. After washing in PBS, samples were incubated for 45 min at room temperature with anti-rabbit Alexa-Fluor-488- and anti-mouse Alexa-Fluor-555-labeled secondary antibodies (ThermoFisher Scientific, #A11008 and #A211422, respectively).