Fig. 5

KPL-404 is not internalized upon binding to CD40 on primary B cells. HD PBMCs were stained with FVD and anti-CD19-Alexa Fluor 488, anti-CD40 KPL-404-AL647 or G28-5-AL647, or anti-CD22-AL647 control (1 μg/ml) and analyzed by imaging flow cytometry. Gated live CD19⁺ B cells (gating strategy is presented in Fig. S6) comprising 400–500 B cell images were analyzed for Ab internalization at 4 °C, 0.1% sodium azide (non-permissive conditions) or after incubation at 37 °C for 1 h (permissive conditions). a–d Representative images of B cell analysis. (a) KPL-404-AL647 binding and internalization; (b) CD19-Alexa Fluor 488 and KPL-404-AL647 binding and internalization, showing CD19 and KPL-404 signal overlay; (c) anti-CD22-AL647 binding and internalization; and (d) G28-25-AL647 binding and internalization under non-permissive (4 °C) or permissive conditions (37 °C). e Cumulative data of three independent experiments. Graph depicts the ratios of the internal mask MFI and membrane mask MFI as a measure of internalization