I am attempting to design a high throughput FACS based phagocytosis assay to investigate the effects of a couple of different drugs on the rate of phagocytosis in a stimulated immortalized microglia like cell line. I know the standard of the field for the FACS based assays is to conjugate the substrate that people hope to be phagocytosed to a PhRodo dye to ensure that the signal is composed of only internalized particles versus objects which are just lodged in the membrane, but I am not sure how the dye would interact with the conjugated protein. Does anybody have any experience with these molecules? Any tips or concerns? Thank you in advance!
Kieona Cook, mostly, dyes are conjugated to lysine residues via activated ester chemistries (usually N-hydroxy succinimide + carbodiimides), with one to a few dye molecules per protein molecule. The modified locations usually are more or less random, depending on solvent accessibility. So I wouldn't expect dramatic differences and just give it a try. If you have access to fluorescence microscopy, you might obtain some informative slides.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining