Phosphorylation of Syk and PLC-γ2 in BCR-stimulated PBMCs

PBMCs stained with mAbs against CD19, CD20, CD3, CD27, CD21, IgD, and BV421-conjugated RBD-B.1 were resuspended in RPMI 1640–10% FBS and stimulated with anti-BCR as described previously (Kardava et al., 2018 (link)), with the following modifications. The cells were stimulated at 37°C for 2 min with 10 μg/ml goat F(ab’)₂ anti-human IgA/G/M (# 109-006-064 from Jackson ImmunoResearch Laboratories). For the detection of phosphorylated signalling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and stained with Alexa Fluor 488-conjugated mAb against phosphorylated Syk (p-Y348) and Alexa Fluor 647-conjugated mAb against phosphorylated PLC-2 (p-Y759) (BD Biosciences). The samples were acquired on an Aurora cytometer and analysis was performed by FlowJo V10.