ClickTech EdU T Cell Proliferation Kit 647 for Flow Cytometry

Mostly, dilution-based T cell proliferation probes conjugate unspecifically to amine groups in cells. Each cell division then results in dilution (takes several days) of the probes. Main disadvantages are cytotoxicity, an impact on T cell activation and long incubation time (several days and up to a week) before readout. The superior EdU cell proliferation assay relies on the incorporation of the modified uridine analog EdU in the DNA of proliferating T cells (just 2 hours before harvesting) and subsequent detection. Both methods enable detection and quantification of T cell proliferation by flow cytometry. Our EdU incorporation assay is very sensitive, and can be multiplexed with other fluorescent stainings. 

• EdU outperforms a standard dilution-based probe in detecting T cell proliferation 
• The EdU assay is less cytotoxic for human T cells 
• The EdU assay offers superior signal-to-background ratio 
• The EdU assay allows for better discernable interferon gamma responses 

Yes, this is feasible. Please note that staining with PerCP, APC and APC-based tandems may be performed before the EdU detection step (click reaction), while RPE, PR-tandem or Quantum Dot antibody conjugates and detection of intracellular antigens should be performed after the click reaction. Check also the user manual for further information. Please, make sure that the emission spectra of the fluorochromes to be combined do not overlap extensively, so that their signals can be distinguished unequivocally using appropriate emission filters or spectral demixing. 

It is possible to safely take a break after the fixation step. Remove the fixation solution, wash as suggested in the user manual and store the cells in wash buffer at 4° C. The permeabilization step is not required immediately. However, the protocol may also be safely paused after permeabilization, following the same recommendation as above. 
It is important to not pause the protocol if the click cocktail for EdU detection has been prepared, since the cocktail reacts optimally within 15 minutes. 

The duration of the EdU incubation step may need to be adjusted according to your cell type or organism, the proliferative state of the cells (e.g., quiescence, lymphocyte activation), the EdU concentration applied and even the experimental design. For a start it is advisable to refer to a published protocol (which is close to your experimental setup) and to test the conditions with a low number of samples. As a general guideline we recommend to use a maximal EdU concentration in cell culture medium of 10 µM for incubations of a few hours to maximum one day. For longer incubation durations the concentration should be decreased to 1-5 µM. Cells that divide rapidly (once a day) generally require shorter incubation compared to cells with lower proliferation rates. Unless specific T lymphocyte preparations are expected to be in a rather quiescent state, a 2 h pulse with 10 µM EdU is usually appropriate for activated human T lymphocytes (and likely mouse ones as well).