Submicron bead calibration kit

Polystyrene (PS) bead standards (200, 500, and 800 nm in diameter) with refractive index ɳ = 1.59 and Yellow-Green fluorescent label were obtained from Polysciences Inc. (Submicron Bead Calibration kit, catalog number BLI832, Bangs Laboratories Inc.). A bead standard mixture of Yellow-Green fluorescent PS beads with refractive index ɳ = 1.59 (110 and 500 nm in diameter) and non-fluorescent silica beads with refractive index ɳ = 1.43 (180, 240, 300, 585, 880, and 1300 nm in diameter) was obtained from Apogee Flow Systems (product #1493). Unlabeled PS beads (2.0 µm in diameter) were obtained from Spherotech Inc. (catalog number PP-20-10). CountBright absolute counting beads were obtained from Invitrogen (catalog number C36950). Quantum Alexa Fluor 488 MESF calibration bead kit was obtained from Bangs Laboratories, Inc. (catalog number 488).
The following dyes were obtained from Invitrogen/Invitrogen: Hoechst 33342 (catalog number H3570), FM4-64 (catalog number T13320), calcein AM (catalog number C34852), calcein red-orange AM (catalog number C34851), LIVE/DEAD Fixable Violet amine-reactive dye (catalog number L34955), and MitoTracker Deep Red FM (catalog number M22426). Antibodies used in these studies and their concentrations are summarized in Table 3.

Up to 2 µg of lEVs were blocked for 15 min at RT in 20 µl PBS + 1% EV-depleted FCS and stained with fluorescently-labeled antibodies directed against EpCAM (#324208, 30 ng/sample), ROR1 (#357803, 2.5 ng/sample, both from Biolegend), ROR2 (#FAB20641G, 12.5 ng/sample, R&D systems), or corresponding isotype controls at the same concentration (#400321 and #400113 from Biolegend, #IC003G from R&D systems) for 20 min at RT. Fluorescence was recorded on the FACSymphony A1 (BD) flow cytometer and the percentage of positive events in the lEV gate was determined in relation to the respective isotype control. The submicron bead calibration kit (#832, Bangs Laboratories) was used to define the gate for lEVs. For EV uptake studies, cells were pretreated with Dynasore (12.5 µM) for 2 h prior to the addition of DiR-labeled EVs (10 µg/ml) for 24 h. Cells were washed twice with PBS and the mean fluorescence DiR intensity of single cells was recorded on the FACSymphony A1 (BD) flow cytometer. Data was analyzed with FACS Diva (version 9.0.2, BD) and FlowJo (version 10.6.1, BD) software. Flow cytometer acquisition settings were maintained for all samples, including triggering threshold, voltages, and flow rate.

Quantification of EV was carried out with the FACSAriaIIIu (BD Biosciences, Heidelberg, Germany). Submicron Bead Calibration Kit (0.2/0.5/0.76 μm; Bangs Laboratories, Fishers, IN, USA) and Flow Cytometry Size Calibration Kit (1/2 μm; Thermo Fisher Scientific, Waltham, MA, USA) were used to define the counting gate up to 1 μm. Sure Count Particle Standard 3 μm (Bangs Laboratories) were placed within the plot area for subsequent quantification of EV. Additionally, 0.1 μm filtered PBS and Count Particles diluted in 0.1 μm filtered PBS were evaluated for contamination with submicron particles.
To evaluate the impact of “swarm detection” of small particles in FACS [27 (link)] we additionally employed NanoSight measurements for EV concentration and size (NanoSight LM 14, Malvern Instruments GmbH, Herrenberg, Germany). Data were acquired in 10 repeats for 10 sec each per EV sample (n = 6).