Hello everyone! I have started a primary cell culture from murine heart, bladder and tight. I need to do a proliferation assay using EdU-Click kit, and I was wondering how long should I incubate the cells with the EdU. I optimized the protocol on AsPc-1 (pancreatic cancer) and PS-1 ( satellite cells) cell lines, and incubation of 1h was fine, as the cells replicate quite fast. But with primary cells I guess the situation is a bit different, they replicate slower, so I guess 1h is not enough. Has someone used Edu kit with primary cells? Do you have any suggestions?
Thank you in advance!
Hello Elisabeth,
Here is an example, where primary cultures of mouse ventricular cardiomyocytes were labeled by 2 μM EdU for 12 h:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960126/
In general, it's a balance between EdU concentration and length of incubation time - higher EdU concentrations allow a shorter incubation time while lower EdU concentrations for a longer incubation time.
I suggest using a concentration of 10 µM for 1-2 hours as a starting point.
Do you want to do a pulse labelling experiment or monitor the synthesis of new DNA over a longer time?
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