Figure 3.

Dara is internalized into primary CD38⁺ MM cells in the context of the total bone marrow microenvironment. Total cellular fraction isolated from BM of a Dara naïve MM patient was treated with 100 µg/ml of Dara or Ctrl IgG and incubated at 37°C or at 4°C for 2 hrs. Cells were then washed with PBS1X and treated with FITC conjugated anti-human IgG for detecting surface Dara/CD38 complex, and TRITC conjugated anti-human IgG was used to evaluate Dara/CD38 or non-specific IgG/CD38 complex internalization. Cells were also stained with CD138 APC to label CD138+ MM-PCs among the total cellular fraction and analyzed by Flowsight cytometric analysis.(A-B) LIVE cells gated as in Sup. Fig. S4 were evaluated for CD138 expression. CD138+ and CD138+/CD38+ cells were evaluated for CD38 internalization after incubation with Dara at 4°C (A) and at 37°C (B); (C-D) LIVE cells gated as in Sup. Fig. S4 were evaluated for CD138 expression. CD138+ cells were evaluated for CD38 internalization after incubation with Ctrl IgG at 4°C (C) and at 37°C(D); (E) Representative images of cells gated in the B panels as Intern.+ showing bright field (CH01), Dara/CD38 complex on the membrane (CH02), internalized Dara/CD38 complex (CH04) and then merged images from both channels. For improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively.