T12 icorr transmission electron microscopy

The invasion assay was performed in transwell assay chambers with 8-μm pore size (Corning Inc., Corning, NY, USA) and ECM (Sigma Aldrich, USA) coating as previously described [17] (link). A549 and H1299 cells were added separately at a density of 5 x 10 4 cells in the upper chamber of each transwell together with 20 µg normoxia or hypoxia-derived A549 EVs in serum-free DMEM. The lower chamber contained 10% FBS-DMEM. After 24 h, cells that remained in the upper chamber was removed with cotton swab soaked in PBS. Cells that invaded into the lower chamber were xed in 100% methanol and then stained with 0.1% crystal violet for cell counting with a bright eld microscope at 10x magni cation.
Transmission electron microscopy A549-derived EVs were 20-fold diluted in PBS and a 7 µl volume added onto a glow discharged carboncoated grid and incubated for 1 min. Thereafter, 2% uranyl acetate was added to the sample and incubated for 1 min. Excess uranyl acetate was blotted off with lter paper. The grid was air-dried for 10 min and subsequently imaged using the T12 Icorr transmission electron microscopy (TEM) at 120 kV (FEI Company, USA).