Bodipy tr ceramide

The exosomes were labeled with BODIPY TR ceramide (Thermo Fisher Scientific, United States) and then resuspended in 10% exosome-depleted FBS-ECM, added to HUVECs at 80% confluence, incubated for 4 h, and imaged under fluorescence microscope.

BODIPY® TR ceramide (Thermo Fisher Scientific, Waltham, MA, USA) diluted in DMSO (1 mM solution) was used for in vivo labeling of exosomes. DPCs were cultured in standard conditions and the dye solution was added to the flask (1 µL/mL). After 20 min of incubation, the medium was removed, the cells were washed with PBS three times and fresh medium containing exosome-depleted FBS (Invitrogen) was added. The cells were cultured for another 48 h. Afterwards labeled exosomes were isolated from the cell medium using the total exosome isolation agent (Invitrogen) as previously described.
HBMMSCs were cultured in an 8-well Chamber Slide System (Ibidi, Planegg, Germany). The fluorescently-labeled exosomes were added to HBMMSCs. After 3 h of incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA), and stained with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific). After washing in PBS, the chambered coverslip was removed, and the samples were mounted in ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology). The samples were analyzed with a fluorescence microscope (BX60; Olympus, Tokyo, Japan). For the negative control, only cells without labeled exosomes were used.

Fresh erythrocytes (1% hematocrit) were washed in complete RPMI-Hepes culture medium. Bodipy TR Ceramide (Thermo Fisher Scientific) was added at 1:1000 dilution and incubated for >1 hr. Erythrocytes were washed 2–3 times with complete RPMI-Hepes and then resuspended in 2 mL of complete RPMI-Hepes. Highly synchronous schizonts (>5% parasitemia from a ~3% hematocrit 30 mL culture) were magnet purified, then added to the labelled erythrocytes (0.1% haematocrit) and 2 mL of this transferred to a 35 mm Fluorodish (World Precision Instruments). Live imaging was performed at 37°C on a Leica SP8 confocal microscope. A 63x/1.4 NA Oil Immersion objective on the Leica SP8 confocal. Time-lapsed images were collected every second upon schizont rupture (594 filter) with an 8 kHz resonant scanner with 4x line averaging and HyD detectors. Cells were maintained at 37°C in a low O₂ and CO₂ nitrogen atmosphere. ImageJ Fiji was used to assemble image series and perform image analyses. Deformation scores were determined according to an established method (Weiss et al., 2015 (link)).

Neural crest cultures were incubated with BODIPY TR Ceramide (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cultures were incubated with 5 µM BODIPY ceramide-BSA (Thermo Fisher Scientific) diluted in cold Hanks’ balanced salt solution (HBSS) for 15 min at 4°C before washing with cold HBSS and re-incubating at 37°C for 2 h prior to imaging. Hoechst 33342 (Thermo Fisher Scientific) was added to HBSS at a concentration of 1 μg/ml and cultures were re-incubated at 37°C for 10 min, washed with HBSS, and then fresh medium was added prior to imaging. SYTO14 Green (Thermo Fisher Scientific) or WGA CF488A or CF633 (Biotium) was added to neural crest medium at a concentration of 2.5–5 µM for 10 min at 37°C. Cells were then washed with PBS, and fresh medium was added prior to imaging.

Processing body (PB) and exosome were visualized with EGFP-Dcp1 and CD-63-GFP (System Biosciences, Palo Alto, CA) plasmids, respectively, which were transfected in COS7 cells using FuGENE6 (Promega, Madison, WI) following the manufacturer’s protocol. The Golgi apparatus was visualized with BODIPY TR ceramide complexed to BSA (Thermo Fisher Scientific). In cells in which either exosome or Golgi apparatus was visualized, fluorescence images of miRNA and organelles were taken 60 min or more after pre-miRNAs were microinjected. Actin was visualized with phalloidin, tetramethylrhodamine B isothiocyanate (Sigma Aldrich, St. Louis, MO), which was resuspended in DEPC-treated water (Thermo Fisher Scientific) and microinjected into COS7 cells. Microtubules were visualized with CellLight Tubulin-GFP, BacMam 2.0 (Thermo Fisher Scientific).