Journal: Oncotarget
Article Title: CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro
doi: 10.18632/oncotarget.8864
Figure Lengend Snippet: ( A ) Indicated cells (4 × 10 6 /mL) were cultivated for 2 h in serum-free medium. Under these conditions they showed no loss of viability. The cell supernatant was precleared by a sequence of centrifugation steps before it was subjected to ultracentrifugation for 2 h at 100000 × g. The pellet was suspended in 1 ml of PBS and tested by nanoparticle tracking analysis (NTA). The graph shows an overlay of 5 independent determinations of the L540 EVs. The mean diameters of EVs from all tested cell lines are shown in the summarizing graph. ( B ) The EVs from 8 × 10 7 KM-H2, L1236 or L540 cells were immobilized at 4.5 μm-microspheres. Aliquots of the microspheres were incubated with ADAM10 antibody (red line) or isotype control (filled histogram). The beads were labeled with fluorescence-labeled anti-mouse IgG and evaluated by flow cytometry. ( C ) ADAM10 was determined by Western blotting in Triton X-100 lysate from 1 × 10 5 L540 cells (cells) or EVs from 8 × 10 7 L540, KM-H2 (KM) or L1236 (L12). ( D and E ) Aliquots of EVs from 4 × 10 7 cells or 200 ng of recombinant ADAM10 or ADAM17 were suspended in 25 mM Tris-HCl, pH 8 containing 6 × 10 −4 % Brij-35 in the presence or absence of 3 μM BB3644 or 3 μM GI254023X. Then, the aliquots were incubated with the fluorescent substrates PEPDAB010 (D) or PEPMCA001 (E) (BioZyme Inc., Apex, NC) in black microtiter plates at 37°C. Fluorescence was determined in a kinetic study at 530 nm (D) or 393 nm (E) as indicated. The data show means of two independent experiments minus background fluorescence without EVs.
Article Snippet: The fluorescence resonance energy transfer metalloproteinase substrates PEPDAB010 and PEPMCA001 were from BioZyme Inc. (Apex, NC).
Techniques: Sequencing, Centrifugation, Incubation, Control, Labeling, Fluorescence, Flow Cytometry, Western Blot, Recombinant
