Table 1.
Fluorescent label, antibodies, isotype controls and secondary antibody used for flow cytometry and Western blot analysis.
| Antibody/dye | Concentration or dilution | Antigen | EV specificity | Source |
|---|---|---|---|---|
| Flow cytometry | ||||
| Bio-maleimide (BODIPY FL N-(2-aminoethyl)-maleimide) | 0.5 μM | Thiol groups | All EV | Molecular probes |
| aAnti PLAP-PE (NDOG2) | 0.5 μg/ml | PLAP | STBEV | In-house antibody |
| IgG1-PE (clone MOPC-21) | 0.5 μg/ml | Isotype control | Biolegend | |
| Anti HLA class I-Alexa 647 (clone W6/32) | 2.5 μg/ml | HLA class I | All EV except STB and RBC derived | AbD Serotec |
| IgG2a-Alexa 647 (clone MRC OX-34) | 2.5 μg/ml | Isotype control | AbD Serotec | |
| Anti CD41-PECy7 (clone P2) | 0.25 μg/ml | CD41 | Platelet EV | Beckman Coulter |
| IgG1-PECy7 (clone MOPC-21) | 0.25 μg/ml | Isotype control | Beckman Coulter | |
| Anti CD235a/b-PECy5 (clone HIR2) | 0.05 μg/ml | CD235a/b | RBC EV | Biolegend |
| IgG2b-PECy5 (clone MPC-11) | 0.05 μg/ml | Isotype control | Biolegend | |
| Western blotting | ||||
| Anti PLAP (NDOG2) | 1 μg/ml | PLAP | STBEV | In-house antibody |
| Anti Alix (clone 3A9) | 1/1000 | Alix | Exosomes | Cell signaling technology |
| Anti CD63 (clone MEM-259) | 1 μg/ml | CD63 | Exosomes | Abcam |
| Anti CD41 (clone 745201) | 2 μg/ml | CD41 | Platelet EV | R&D systems |
| Anti CD235a/b (clone HIR2) | 0.5 μg/ml | CD235a/b | RBC EV | Santa-Cruz |
| Anti CD45 (clones 2BII + PD7/26) | 0.375 μg/ml | CD45 | Leucocyte EV | Dako |
| Polyclonal goat anti-mouse immunoglobulin HRP | 0.25 μg/ml | Mouse immunoglobulins | Dako | |
a
In-house antibody, custom conjugation by Biolegend.