Phycoerythrin pe

3(hydroxy-4-nitrophenyl)acetyl (NP) (BioSource) was coupled to phycoerythrin (PE), chicken gamma globulin (CGG) or streptavidin (all from Sigma) as we previously described [29 (link)]. Biotinylated I-Ealpha peptide was synthesized by Invitrogen and used to generate both NP-streptavidin-I-Ealpha and NP-streptavidin-AF647-I-Ealpha which were prepared and tested in control experiments as described [28 (link)].

The surface markers of the PDE-EVs (tetraspanins, CD9, CD63, and CD81) as well as annexin positivity and calnexin negativity were determined by flow cytometry using FACS Calibur (BD Biosciences, San Jose, CA, USA). Thirty microliters of 1.5 × 10¹¹ particles/mL PDE-EVs was first adsorbed onto the surface of formaldehyde/sulfate Latex-Beads (Molecular Probes, Eugene, OR, USA). In brief, EVs were loaded onto 3.8 μm diameter 4.0 v/v% beads dissolved in 100 μL phosphate-buffered saline (PBS) and incubated overnight at 4 °C under gentle agitation. PBS containing 2% BSA was used as a negative control. Samples were incubated for 1 h in PBS containing 2% BSA; then, 300 μL 0.9% NaCl solution was added, and finally, the samples were centrifuged for 10 min at 400× g. The beads were resuspended in 200 μL 100 mM glycerol solution and incubated for 30 min at RT. Samples were then labeled with specific antibodies against CD9 ((fluorescein (FITC), Sigma;#:SAB4700095)), CD63 ((phycoerythrin (PE), Sigma;#:SAB4700218)), CD81 (FITC, Molecular Probes;#:A15753), annexin V (FITC, SONY; #:3804530), and calnexin (E-10, Santa Cruz Biotechnology, Dallas, USA) for 24 h at 4 °C. For data analysis, FlowJo Software v9(Tri Star Inc., Ashland, OR, USA) was used.