I am using the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) to measure proliferation on human adipose-derived mesenchymal stem cells.
https://www.thermofisher.com/order/catalog/product/C10420?ICID=search-product
I would like advice on the following:
1. The recommended EdU concentration is 10 μM, however, this appears to be too low. What is the ideal concentration to use for mesenchymal stem cells?
2. At what time points do you run the assay to measure proliferation?
Thanks in advance for your response.
1. We used 10μM. It worked well. You just consider that 1mol=6.02*10e23 molecular, 10μmol=6.02*10e18 molecular. There will be 6.02*10e15 per ml.
For this assay, EdU is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry; a copper-catalyzed covalent reaction between an azide and an alkyne. The alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ® 488. The advantage of EdU labeling is that the small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions. Compare with BrdU assay, which needs to do DNA denaturation; the EdU assay is much better and simpler.
2. The time point must be the cells have DNA synthesized, so the EdU can incorporated into cells. We did the detection after five-days EdU added for human BM-MSC. We can see the clear proliferation peak.
Hello
Look at links here
Good Luck
https://tools.thermofisher.com/content/sfs/posters/CYTO-2010_Analysis_of_hMSCs_by_flow_and_Imaging.pdf
https://stemcellres.biomedcentral.com/articles/10.1186/s13287-016-0367-3
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