Has anyone tried to study Wnt signaling in HEK293T cells? I have been trying to measure the Wnt activity using TOP/FOP-FLASH assay but the readings that I got are rather similar in both TOP and FOP, I couldn't see a high TOP/FOP ratio. I also tried to activate the Wnt activity using CHIR99021 but I did not see any activation. Can anyone shed me some light? Below is my culture condition:
TOP or FOP plasmid + Opti-MEM and Lipofectamine + Opti-MEM are incubated at room temp for 5 min respectively. Both solutions are then mixed and incubated for another 15 min. Then, the mixture is equally distributed into each well (I used a 6-well plate). Then, 0.5M of HEK293T cells are added into each well (1ug plasmid in each well too). After 6hrs, new media supplemented with 5uM CHIR are added. Luciferase assay was performed after 24hrs.
Did you have a positive control to demonstrate there is a successful transfection?
ALEX CHUN CHEUNG I don't have a transfection control, what would you recommend? I was able to detect high Wnt activity, so I suppose the transfection works fine?
Yen Teng Tai
I don’t get your words.
“......measure the Wnt activity using TOP/FOP-FLASH assay but the readings that I got are rather similar in both TOP and FOP, I couldn't see a high TOP/FOP ratio. I also tried to activate the Wnt activity using CHIR99021 but I did not see any activation.”
From this, what i get is that you fail to detect any wht signal, even in the presence of CHIR99021.
You may need a positive control by transfecting a plasmid with a known reporter gene.
https://altogen.com/transfection-controls-need-include-experiment/
or have u tested a range of different concentration of CHIR99021"?
or have u tried different activators?
Here I attached the result that I got. Both TOP and FOP show almost equal readings. I have tried different concentrations of CHIR and even included Wnt3A as control but still, none of them work.
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