Imaging Flow Cytometric Analysis of EV Uptake

BMDMs were incubated for 24 hr with CellVue-labeled H. polygyrus- or RAW264.7-derived EVs, and then they were stained with 50 nM LysoTracker Green DND-26 (Cell Signaling Technology) for 60 min at 37°C. At least 10,000 live-cell images for each sample were acquired using a six-channel ImageStream^X Mark II (EMD Millipore) imaging flow cytometer equipped with 405-, 488-, and 642-nm lasers. Samples and single-color compensation controls were acquired at 40× magnification. Data were analyzed using IDEAS 4.0.735 software (EMD Millipore) for Lysotracker/CellVue Bright Detail Similarity (BDS). BDS is a normalized measurement of co-localization or overlap between the fluorescent signals and pixel intensity, as described elsewhere (Phadwal et al., 2012 (link)).