Inhibition of Btk phosphorylation in CD19+ B cells from CML patients on TKI coincubated with autologous plasma. Cryopreserved PBMCs from CML patients on TKI (imatinib, n = 3; nilotinib, n = 3; and dasatinib, n = 3) were thawed, washed, and cocultured with autologous plasma or RPMI/10% fetal calf serum overnight. PBMCs were then stimulated with 5 mL of 50 mM of H₂O₂ for 15 minutes at 37°C. The stimulation was terminated by the addition of 5 mL prewarmed Cytofix Buffer (BD Biosciences, San Jose, CA) at 37°C for 12 minutes. Cells were fixed/permeabilized and stained with pBtk-PE (BD Biosciences) and APC-conjugated anti-CD19 (BD Biosciences). Data acquisition was performed on the FACSCalibur, and FlowJo software was used for analysis. MFI of Btk phosphorylation following incubation with autologous plasma in gated CD19+ B cells from representative CML patients on imatinib, dasatinib, or nilotinib is presented (right panel).