I am trying to set up a phagocytosis assay using CFSE-labelled apoptotic thymocytes as particles. I want to quench external fluorescence to make sure my FACS measurements really reflect phagocytosis, and not particle binding on the surface of macrophages. Can anyone help?
Trypan blue can be used to quench extracellular fluorescein but CFSE labels intracellular proteins. Therefore I think it is unlikely that you will be able to quench the CFSE from the labeled thymocytes which are on the surface but not yet internalised. Have you tried any of the commercial phagocytosis assays which uses fluorescein labeled particles which can be quenched with trypan blue to distinguish adhesion from internalisation?
Thanks for your response. In fact, I want to specifically measure phagocytosis of apoptotic bodies. Would you have a protocol for the use of trypan blue?
Here is reference to protocol by Elizabeth Simons in Current Protocols in Cytometry:
Simons, E.R. Measurement of phagocytosis and of the phagosomal environment in polymorphonuclear phagocytes by flow cytometry. Current Protocols in Cytometry. Chapter 9. Unit 9.31 (2010).
Good luck.
- Badges
- Science method