PKH67 EVs staining and Immunofluorescence
AnEVs were stained with the green fluorescent membrane dye PKH67 accordingly to manufacture instructions (Sigma). To evaluate the interaction between EVs from anaphylactic patients and endothelial cells (EC) in vitro confocal microscopy was performed. HMVEC-L were seeded in P8 chambers (IBIDI) and incubated with PKH67-EVs during 5h. Next, cells were fixed and stained with Texas Red-X phalloidin and DAPI. Images were acquired in an inverted confocal microscope (LSM700; Carl Zeiss, Oberkochen, Germany).