Improved click chemistry demonstrating EdU cell proliferation with GFP expressing cells and R-PE based immunophenotyping

The current Click-iT® EdU kit, for measuring cell proliferation, cannot be use for the simultaneous detection of EdU, GFP and R-PE fluorescence due to the presence of copper and reactive oxygen species mediated damage to fluorescent proteins. We present modifications to the click reaction resulting in“copper safe” catalysis of the click reaction. Flow cytometry measurements of cell proliferation using Click-iT® EdU are demonstrated to be compatible with immunophenotyping panels using R-PE conjugates and GFP expressing proteins. The basis of the click chemistry improvement is the use of a copper (I) ligand combined with a modified dye azide detection reagent. We optimized components in the improved click reaction which best preserved R-PE fluorescence while obtaining a bright EdU signal. Jurkat cells were pulsed with EdU, labelled with PE conjugated antibodies and then fixed and permeabilized. EdU incorporation was then detected using click chemistry, demonstrating improved detection while maintaining PE signal. Additionally, BacMam transduced cells expressing GFP were treated with cell cycle inhibitors then pulsed with EdU and used to demonstrate GFP and click chemistry compatibility. The modified click reaction is an improvement over the original copper based click reactions and will further enhance the utility of EdU based cell proliferation applications in flow cytometry.