Stock solutions of MEK/ERK inhibitor - PD0325901 and GSK3 inhibitor - CHIR99021 were prepared in DMSO and stored as aliquots in tightly sealed eppendorfs. For how long can these aliquots be stored in such sealed eppendorfs at -20C. I have had them for 6 months.
I am using them for murine ESC culturing in N2B27+2i media on gelatin coated plates. What are the signs to look for in the culture when the activity of these two inhibitors is sub-optimal ?
Thanks in advance for the help.
If the -20 didn't suffer any freeze-thaw cycles, they are stable for a long time, even more than a year. In -80 for backup stock can be good too.
If you want to check that your stock is still properly active, you can test in the following way:
- Rex1-dGFP reporter mouse ESCs
- grow in 2iLIF vs 2i vs PD+LIF vs CH+LIF vs single components for a few passages
- profile by flow cytometry
Since all two-component combinations 2i, PD+LIF and CH+LIF should maintain the naive dGFP+ status at >98% (if your N2B27 base is good!), but single use of PD, LIF or CH would not, this is a clear diagnostic. Whereas, in 2iLIF, if one component is not working properly, it will be masked by the other two allowing self-renewal of naive state anyway.
Hope that's clear!
Dear Hannah T Stuart
Thank you for the suggestion. I tried FACS with only one passage in said conditions. The profiles were tight unimodal (representative figure titled PD_LIF) for all the two-component combinations except for 2i. The 2i sample had heterogeneity (figure titled 2i_culture). The base media seems to be good as the entire population irreversibly exit naive state by 48hr culture in N2B27 and I saw minimal cell death during prolonged culture in N2B27.
Looking at the profiles, do you think it warrants the preparation of new stock of two inhibitors or is the population profile acceptable?
I recently started using new aliquots from the same batch of PD and Chiron stocks which have been stored at -80C and will repeat the experiment again. I will let you know the outcome in any case.
Once again thanks very much for the suggestions.
Regards
For our PD0325901 and CHIR99021, our chemists usually recommend that if they have been stored in aliquots, at -20°C in tightly sealed vials they will generally be useable for up to one month. However, if you are going to use them after this, you should allow them to equilibrate to RT for at least one hour before opening. You also need to ensure that they remain fully solublised in solution before use, as they can drop out of solution during thawing (and if they are not solubilised properly, this will obviously affect the activity).
Samantha Roome
Thanks for the valuable information. I generally use the entirety of aliquots at once. But for the future, I will make sure that aliquots are used within one month.
Regards
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