Hi all,
I produced WNT3a conditioned medium starting from
L Wnt-3A (ATCC® CRL-2647™), but when I perform the TOP FOP assay I only have a 2X increase compared to not treated.
I followed both ATCC and Clevers' protocol and I also tried to keep cells in culture for more days but nothing has changed.
I need it for intestinal organoids culture and I read you need to have at least 5X increase.
Does anyone have any suggestion?
Hi, did you use a positive control (e.g. recombinant Wnt3a) for the TOP FOP assay? Did you try different FBS batches or different FBS suppliers?
Many cells are sensitive to O2 levels. A great deal of literature shows that cells grown in physiologic culture often have very different growth and expression profiles. This can be tested by growing in the Petaka G3 cell culture cassette, which automatically regulates O2 levels to physiologic (3-6%) versus what you are doing now (21%). So, just do a quick test...et some Petaka G3 (LOT for adherent or FLAT for suspension), grow in the same media for the proper time and see if your cells are not producing now. You may see huge improvements with this profound improvement to your culture conditions.
www.celartia.com
Agnieszka Pastula yes I use recombinant WNT3a as positive control and I also tried different FBS suppliers, but nothing changed
Be careful about the number of passages of the cells and how they were maintained before you use them. Better to buy a new batch that you are sure they were cultured properly without over splitting
Hello Pamela,
Using the right selection antibiotic?
1. The recommended selection antibiotic and concentration for producing conditioned media with L Wnt-3A (ATCC® CRL-2647™) is 0.4 mg/ml G-418. However I believe that the Clevers lab. use a slightly different cell line that requires Zeocin.
2. I would suggest keeping the cells growing on selection medium for a week before attempting to make conditioned medium; to make sure you have a pure colony of cells.
Filtering correctly?
1. After you have made your conditioned medium, make sure to use a low-protein binding filter cups to avoid loss of Wnt-3a. I recommend testing a before and after filteration to make sure the issue is not with your filter.
Storing the Wnt-3a medium correctly before testing?
1. Although, recommended storage temperature for Wnt-3a is 4oC for 6 months, I have tested batches stored at -20 or -80 and have not seen a significant decrease in activity using the TOP/FOP assay.
Finally, you may have seen the following paper: https://www.nature.com/articles/nprot.2016.097 - they have written a robust method for production of Wnt-3a conditioned medium.
I hope this helps. Let us know how you get on please. Thank you,
Salman.
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