fig3

Figure 3. Characterization of CD9 and CD81 EVs by SP-IRIS. (A) Sizes measured by interferometry are in the range ascribed to small exosomes; (B) Heat map shows that RANK was only detected in CD9-captured EVs from osteoclast-precursors and osteoclasts. Samples were diluted 10X or 2X. Numbers are particle count × 10³; (C) EVs captured by anti-CD9 antibody from 2X diluted sample and detected by interferometry (Total), anti-RANK antibody, anti-CD81 antibody, or anti-CD9 antibody. Note that not every EV captured by anti-CD9 antibody is expected to be detected by a second anti-CD9 antibody as antigen may be patched on the capture side of EVs; (D) Very few EVs were captured by CD9 control antibody; (E) EVs captured by Anti-CD81 from 2X diluted samples. CD81 EVs are more common in 4T1 cells than osteoclasts or their precursors, and regardless of cell source, RANK was not detected on EVs captured with an anti-CD81 antibody; (F) Very few EVs were captured by CD81 control antibody. The asterisks in panels C and E indicate significant differences from controls; the n = 3. The results indicate statistics performed first by ANOVA, then by t-tests, P < 0.05; *: P < 0.05.