Direct TIRF imaging of exosome secretion dependent on Munc13-4. (A) pH-sensitive pHluorin (pHl) was inserted into loop 1 of CD63, which localizes to both the limiting membrane and ILVs of MVBs. Initially quenched at the low pH in the MVB, CD63-pHluorin increases fluorescence upon fusion of MVBs with the plasma membrane. (B) MDA-MB-231 cells stably expressing control shRNA (Ctrl, upper row) or Munc13-4 shRNA (Munc13-4 KD, lower row) were transfected with CD63-pHluorin plasmid, stimulated with 1.25 µM ionomycin (at zero time), and imaged for 15 min by TIRF microscopy. The surface of single cells is shown. In the upper row, ionomycin treatment elicited the release of brightened CD63-pHluorin–containing ILVs released into TIRF field. Bar, 5 µm. Inset shows one of the MVBs releasing ILVs as exosomes. Bar, 1 µm. See Videos 2 and 3. (C) Intensity of CD63-pHluorin signal is quantified as fold increase over initial fluorescent value reported as mean values ± SE (≥9 cells/group; *, P < 0.05).