ClickTech EdU Cell Proliferation Kit 555 for HTS

No, at the moment the kit needs live cells to incorporate the EdU, but the detection reaction must be performed on fixed and permeabilized samples. The click cocktail is cell impermeant.

All three methods enable to determine cell proliferation directly, by incorporation of a metabolite analogue and subsequent detection. The 3H-thymidine incorporation assay is very sensitive, but the radioactive compound requires specialized equipment and dedicated lab space for handling. EdU and BrdU assays are non-radioactive alternatives with decreased risk for health and environment. Compared to the BrdU incorporation assay the EdU assay is more sensitive, requires less handling time and needs no harsh DNA denaturing conditions for detection. Therefore, the EdU cell proliferation is also compatible with multiplexing.

Yes, this is feasible. Please note that DAPI staining should be done after the click detection step. Alternatively, SYBR Green DNA staining can be used. But, please note that SYBR green is not compatible with the 488 EdU kits.

It is possible to safely stop the protocol after the fixation step, remove the fixation solution and wash as suggested by the user manual, then the cells can be stored in buffer at 4° C, the permeabilization step is not required immediately. The protocol can be safely stopped even after the permeabilization, following the same recommendation as before.
It is important to not stop the protocol if the click cocktail for the detection of the EdU has been prepared, since the cocktail reacts optimally within 15 minutes.

Antibody staining is compatible with EdU cell proliferation detection when antibody detection is done after the click detection step. Please make sure that the detection signals of both detection methods do not overlap due to identical or similar spectral properties of the dyes. Check also the user manual for more information.

The duration of the EdU incubation step depends on your cell type or organism, the applied EdU concentration 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 maximum of 10 µM final EdU in the cell culture medium 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 slow division (once a week).