My lab was using WNT conditioned media for human breast cancer organoids and we recently madea fresh batch of house made conditioned media. However, we do not have old batch to compare it with new ones. How do I perform my TopFLash Assay in this case? How do I QC my media ? Please help.
Hi Lakshmi,
In absence of already working Wnt-CM as a positive control, I would suggest you to test serial dilutions of your freshly prepared Wnt-CM in parallel with serial concentrations of Wnt3a ligand (10ng/mL to 300ng/mL) (i.e.from R&D, Peprotech), or GSK3i CHIR99021 (0.5-3uM).
I don't know what is the efficiency of transfection on breast organds, but try lipofectamine RNAimax transfection or electroporation using the TopFLash constructs Firefly and Renilla (we use 10ng FF + 10ng of Renilla + 40ng empty vector i.e. PCS2 or pCDNA3 for a 96 well transfection in 2D culture). You can find in Addgene these vectors (https://www.addgene.org/24306/), (http://www.addgene.org/collections/luciferase/)
Good luck!
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