Supplementary Methods
Transwell assay using free PKH67 dye
5x103 U87 recipient cells were plated in wells of a12 well plate. The following day, cells
were washed with PBS. Next 10 µl of PKH67 dye was added to 2 ml of diluent C and
this was divided into two fractions A and B. Fraction A was mixed with media alone
while fraction B was mixed with heparin (final heparin concentration 67 µg/ml). Next
fraction A and B were each divided among three wells containing unlabeled cells. Cells
were incubated for 24 hr at 37C and flow cytometry was performed to measure cell
labeling of recipient U87 cells.
Zeta potential measurement
The
zeta
potential
of
U87-derived
EV
in
the
presence
or
absence
of
heparin was determined using the Zetasizer Nano - ZS device (Malvern, UK) at pH 7.5.
EVs were left untreated or were mixed with 100 µg/ml of heparin for 1 h at room
temperature before measurement.
Determining cellular localization of Gli36-EGFRvIII-derived EVs in recipient U87
cells after 3 h incubation.
EVs from Gli36-EGFRvIII cells were labeled with PKH67 dye as before. Next, EVs were
added to unlabeled recipient U87 cells for 30 minutes at room temperature. Bright field
and fluorescence images were acquired. Next cells were trypsinized, washed in PBS, and
replated and allowed to attach for 3 hours before re-examining them for fluorescence.
EV/cell binding assay and internalization assay
EVs derived from Gli36-EGFRvIII were labeled with PKH67 membrane dye. Next EVs
were incubated with PBS (as control) or 100µg/ml heparin for 15 min at RT. Then two
types of incubation scenarios were performed. In the first, EVs were added to U87-MG
grown on coverslips placed in a 12 well plate and the plate was kept at 4ºC to allow
binding but not internalization of EVs. After 30 min, cells were washed to remove
unbound EVs and fixed in 4% formaldehyde in 1 x PBS. In the second incubation
scenario, the EVs and cells were incubated on ice and washed as above, but then they
were switched to 37ºC for 30 min to allow internalization to occur prior to fixation.
Next, samples were analyzed with confocal imaging using a Zeiss LSM 5 Pascal laserscanning confocal microscope (see section Heparin and EV colocalization).
To
determine whether the PKH67 fluorescence was present at the surface or intracellularly,
z-stack images were acquired from the top of cells in 3 µm intervals to the bottom of
cells.