Isolated exosomes displaying CD81 and CD63 positivity but absent for CD9.
CD9 positive control shows no issues with the antibody.
Anti-CD63 beads were used, and FACS gating was based on the beads.
Is CD9 positivity a mandate?
Isolation was performed using a kit.
Hi, depending on the kit you used, not all EV subpopulations will be homogenously precipitated / purified. CD9 is one of the tetraspanins often used for EV characterization, but its presence or absence does not qualify or disqualify a certain EV preparation. I recommend to refer to the MISEV guidelines for further information and important considerations on designing and reporting your experiments: https://pubmed.ncbi.nlm.nih.gov/30637094/
By selecting exosomes/EVs positive for CD63, a bias might have been introduced. If there are EVs positive for CD63 but not CD9, and other EVs are only single positive for CD9 without CD63, this EV enrichment approach might have missed the CD9-EVs and therefore you do not see CD9 in your EV preparation.
I agree with Alexander Otahal in that bead capture might introduce a bias. CD9 expression varies a lot between cell types, so it is worth trying to measure your EVs again using a higher sensitivity flow cytometer, with an antibody primary conjugated to a very bright fluorophore, such as Alex Fluor (AF). Using the same AF for anti-CD9, CD63 & CD81 Abs will remove fluorescence detection sensitivity biases.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column