Lsr 2 special order flow cytometer

The bead/antibody/EV complex was (a) coupled to a fluorescein isothiocyanate (FITC) fluorescent tag that binds to extracellular vesicles (Exo-FITC, Systems Biosciences; Cat #CSFLOWBASICA-1) and a phycoerythrin (PE) fluorescent tag that specifically binds to the astrocyte surface marker, GLAST-PE (Miltenyi Biotec Inc; Cat #130-118-483); and (b) subsequently analyzed by flow cytometry to confirm AEEV capture. The bead/antibody/EV complex was washed three times with 1X BWB and then incubated with 10 μL of Exo-FITC and 2 μL of GLAST-PE in 240 μL of Exosome Stain Buffer for 2 h on ice with gentle flicking every 30 min. The stained complex was washed three times in 1X BWB and resuspended in 500 μL 1X BWB prior to flow cytometry analysis. The flow cytometric data were acquired using a BD LSR II Special Order Flow Cytometer (BD Biosciences, San Jose, CA). Instrument performance was validated using BDTM Cytometer Setup and Tracking (CS&T) beads (BD Biosciences, San Jose, CA). All data were analyzed using FACS DIVA 8.0 software (BD Biosciences). Debris and small particles were excluded by gating out events with low forward scatter. Fig. 1B shows an example of successful EV capture, and Fig. 1C shows an example of AEEV enrichment.