Igg2b apc

Immune capture and bead-based flow cytometry were carried out as described previously (8 (link),24 (link),25 (link)). In short, 10 µg exosomes in 100 µl PBS were incubated with 1 µg biotin-labeled anti-CD63 (Cat: 353018, Biolegend, San Diego, CA, USA) for 2 h at room temperature on a shaker. Next, 10 µl ExoCap Streptavidin magnetic beads (MBL Life Science, Woburn, MA, USA) were added and incubated for another 2 h at room temperature on a shaker. Samples were washed using a magnetic rack and subsequently stained with the following antibodies/isotype controls for 1 h at room temperature on a shaker: CD14-PE (0.5 µg, Cat: 12-0149-42) and IgG1-PE (0.5 µg, Cat: 12-4714-42) (both from eBioscience/Thermofisher Scientific), CD16-APC (0.8 µg, Cat: 36076) and IgG1-APC (0.8 µg, Cat: 400122) (both from Biolegend), CD44v3-APC (10 µl, FAB5088A) and IgG2b-APC (10 µl, IC0041A) (both from R&D, Minneapolis, MN, USA). The stained complexes were washed twice using a magnetic rack and finally resuspended in 300 µl PBS for flow cytometry. Detection was performed using a Gallios flow cytometer with Kaluza 1.0 software (Beckman Coulter, Brea, CA, USA) and 10000 events were acquired. Data are presented as relative fluorescent intensity (RFI) which is the mean fluorescence intensity of the stained sample divided by the mean fluorescence intensity of the corresponding isotype control.

Cells were stained with 0.5 μg antibody (either CD46-PE [8E2] or IgG1κ-PE, Thermo Fisher Scientific; and nectin-4-APC or IgG_2B-APC, R&D Systems), and receptor expression was measured in a MACSQuant Analyzer 10 (Miltenyi Biotec). The ratios of MFIs of receptor antibody versus isotype control were calculated, and graphs generated using GraphPad Prism 6 software.