Surface Ig expression and BCR signaling in primary DLBCLs. Cryopreserved tumor cell suspensions were thawed and viable tumor cells were isolated from a Ficoll Hypaque monolayer. Thereafter, the tumor cell suspensions were over 90% viable by Trypan blue staining. Light microscopy, light scatter analysis at flow cytometry, and cell-surface Ig expression confirmed the presence of a predominant population of tumor cells. (A) Cell-surface Ig expression in primary DLBCLs. Cell-surface IgG (blue) and IgM (purple) were evaluated by flow cytometry. P1 expressed high levels of sIgG whereas P2 and P3 expressed sIgM. P4 expressed lower levels of sIgG and P5 lacked surface Ig expression. Cells stained with an isotype-matched control Ig are shown in gray. (B,C) BCR signaling in primary DLBCLs. Single-cell phospho-flow cytometry was used to assess pSYK352 and pBLNK expression in the absence (green) or presence (red) of BCR crosslinking or BCR crosslinking following R406 treatment (blue). Primary DLBCLs with intact BCR signaling (B) and ineffective BCR signaling (C) are shown. Cells stained with isotype-matched control Ig are shown in gray.