Pacific blue conjugated anti cd3 clone ucht1

Antibodies with the following specificities were purchased from BD Biosciences: pPLCγ2 (Alexa Fluor 488, pY759), pSYK (PE, pY348), pLCK/pLYN (Alexa Fluor 647, pY505). Phosphatase abundance was estimated by SHP-1 antibody (Santa Cruz Biotechnology). Detection of PBMC subsets was achieved with Pacific Blue-conjugated anti-CD3 (clone UCHT1) and PerCPCy5.5 conjugated anti-CD20 (clone H1), and CLL circulating tumor was confirmed for CD5 positivity with PE-Cy7-conjugated anti-CD5 (clone L17F12, all BD Biosciences). Intracellular reactive oxygen species was measured under resting and H₂O₂-stimulated conditions using 2′,7’-dichlorofluorescin diacetate (DCFDA) (Abcam). For assessment of intracellular species, cells were permeabilized following fixation with PFA with cold methanol at −20°C for 10 minutes. Intracellular staining was carried out at 4°C in PBS + 2% FBS. Cells were washed twice with PBS + 2% FBS and analyzed on an LSRII cytometer (BD Biosciences). Analysis was carried out using FlowJo software (TreeStar), Prism (Graphpad), and with custom tools written in R statistical computing language (R project) and MATLAB (Mathworks). Code is available upon request.