Figure 1:

Comparisons of flow cytometry with confocal microscopy imaging for detection of PD-L1 on exosomes isolated from plasma of three HNSCC patients. Exosomes were captured on beads using biotinylated anti-CD63 mAb and were then stained with labeled anti-PD-L1 mAb for detection of PD-L1 by flow cytometry or by confocal microscopy. A. Exosome immobilization for confocal microscopy: 1.The epoxy-silane slide; 2. Exosomes are deposited into wells on the slide; 3. and 4. Proteins on the surface of exosomes interact with epoxy-silane on the slide via their exposed amine-, thiol- and hydroxyl-groups, immobilizing exosomes for confocal imaging. B. Confocal microscopy images of exosomes of the 3 patients. Exosomes captured on beads and immobilized on the epoxy-silane slide were stained with labeled anti-CD63 (green) and anti-PD-L1(red) mAbs (mag x10, upper panel and mag x252, lower panel). Note differences in staining intensities of the exosomes for PD-L1 (low for patient #1, high for patient #2 and intermediate for patient #3). C. Quantification of immunofluorescence signals for the immobilized exosomes of the three HNC patients. Note that the staining intensity for PD-L1 in each patient’s exosomes corresponds to the flow cytometry data (shown in D). D. The RFVs for PD-L1⁺ exosomes detected by flow cytometry are different for each patient (33, 168, 68).