What type of cells can incorporate EdU?
The EdU cell proliferation assay has been applied to many different cell types and organism. Human cancer cell lines like HeLa, HEK, MOLM are arguably among the most used; however, it can also be applied to animals such as mouse, rat, the nematode C. elegans, crickets (Gryllus bimaculatus), chicken (Gallus domesticus) and zebra fish (Danio rerio) or even plants (e.g. Arabidopsis thaliana) can be applied.
Cells that possess a pyrimidine salvage pathway can phosphorylate EdU to the corresponding triphosphate, which allows the host DNA polymerase to incorporate it into the DNA during replication. Thus, making these cells compatible with the EdU assay.
Can I perform EdU cell proliferation detection on living cells?
No, at the moment the kit needs living cells to incorporate the EdU, but the detection reaction must be performed on fixed and permeabilized samples. The click cocktail is cell impermeant.
How does EdU labelling compare to BrdU or the 3H-thymidine incorporation assay?
All three methods are able to determine cell proliferation directly by incorporating a metabolite analogue, which is subsequently detected. The 3H-thymidine incorporation assay is very sensitive, but the radioactive compound requires specialized equipment and dedicated lab space for its handling. EdU and BrdU assays are non-radioactive alternatives with decreased health and environmental risks. Compared to the BrdU incorporation assay, the EdU assay is more sensitive, requires less handling time and needs no harsh DNA denaturing conditions for its detection. Therefore, the EdU cell proliferation is also compatible with multiplexing.
Can I combine DAPI staining and EdU detection?
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.
When can I stop safely the protocol?
It is possible to safely stop the protocol after the fixation step, remove the fixation solution and wash as suggested by the user manual. The cells can be stored in buffer at 4° C because the permeabilization step is not required immediately. The protocol can also be safely stopped 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.
Is antibody staining compatible with the EdU?
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.
What is the right duration of EdU incubation?
The duration of the EdU incubation step depends on your cell type or organism, the applied EdU concentration and even the experimental design. It is advised 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 the incubations time of a few hours to a maximum of one day. For longer incubation duration, EdU’s 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).
