Hi everybody, I am struggling to get the TOP flash assay to work using 293T cells. I have tried adding 10 ug of recombinant Wnt3a as positive control and also using Wnt3a conditioned media but I don't manage to see any difference in the Luciferase signal in what should be my positive control compared to Not Treated cells. I have also added RSPO-1 at different time points but I didn't have any successful data.
I usually transact 293T cells with TOP FLASH plasmid/ Renilla in a 1/10 Ratio.
The day after I transfer cells in a 96well plate and I treat them with the conditioned media for 48 hours. On day 3 I perform the luciferase assay using the dual Glo kit by Promega.
Is there anybody willing to share a detailed protocol with me?
Thanks in advance for any help provided
Hi Giovanna,
just some points to consider:
- you could try using LiCl as an unspecific activator for the wnt-pathway for optimization purposes (much cheaper than recombinant proteins)
- did you serum starve your cells prior to treatment - otherwise your control might also give you quite high signals?
- try to reduce the time after stimulation - if I can recall correctly the pathway is activated quite rapidly (48 hours seem way too long for me!)?
- also the TOP Flash / Renila ratio for transfection is 10/1 right (not the other way around)?
Best regards and good luck with your experiments,
Christian
Hi Christian,
Thank you for the reply. I have never serum starved the negative control, I will do that.
I will also definitive try to use LiCl at different concentration for my positive control and I will also try a time course.
The Flas/Renilla Ratio i used was 1/10.
Thanks again for all your suggestions and I will keep you posted.
Giovanna
Hi Giovanna,
We do routinely this experiment in the lab. Usually, we plate 30K HEK-293T cells per 96 well in 100uL. The day after, transfect a total of 50ng of DNA per well, following these amounts: 35ng PCS2, pCDNA3 or other empty vector, 10ng of Firefly (TOP Flash plasmid) and 5 ng of Renilla, using X-treme Gene9.
24h after, we add the treatments. I would suggest as Christian mentioned not to leave treatments for long time. We do it between 8h to overnight (20h). Try in parallel LiCl, CHIR99021 or GSK3i BIO. You should see the increase. As another control, keep same treatments on 6well plates, harvest cells and perform qRT-PCR of Axin2. That should confirm whether your cells are responding to WNT signaling.
Good luck and if you have further doubts, don't hesitate.
Anchel
Hi, I am struggling in activating Wnt in this system too. I have tried CHIR99021 but I can't get consistent results. Sometimes I able to see induction by CHIR in 18h -24h but sometimes don't, can anyone give me some advice? I did lipofectamine transfection in 12-well plates with 0.5M of cells and 0.8ug of TOPFLASH plasmid in each well.
Hi Yen Teng Tai , are you using WT HEK293T cells? Check that your cell line is not mutated for beta-catenin or APC (happens a lot in CRC lines), which will render no response neither to Wnt or GSK3i.
If problem persist, try WB of total/active beta catenin, and WB of LRP6 (both should be stabilised).
Good luck,
Anchel
Anchel de Jaime Soguero I am using HCT116 cells. Our lab does not have antibodies for B-catenin nor LRP6. Is there any other way for me to confirm? Perhaps I can perform RT-qPCR to look at the Wnt target genes?
Dear Yen, HCT116 are mutated for a phosphorus-site of Beta catenin, thereby have continuously activated the pathway, being less responsive to Wnts and GSK3i. However, I use them and you still see a response upon Wnt activation.
I recommend you to do the experiment in parallel with HEK293T because of two reasons:
1) They are easier to be transfected than HCT116
2) They respond better to Wnt ligands or GSK3i.
In our lab, we usually transfect 50ng of DNA per well of a 96 well plate (in which we plate between 10-30K cells, depending cell line). From that 50ng DNA, 15ng are TOPFlash plasmid (Firefly), 10 ng Renilla, and the rest empty plasmid (PCS2 or pCDNA3). Treat the cells O/N with concentrations between 1-3uM of GSK3i or with Wnt3a.
If you want double confirmation, check by qPCR genes as Axin2 or Lgr5.
Good luck,
Anchel
Anchel de Jaime Soguero thank you so much for your information! I will give it a try!
Anchel de Jaime Soguero
Hello, I recently started using the TOPFlash assay and I want a positive control to make sure the vectors are working. I read several places that LiCl can be used to activate Wnt signaling. I am looking for an efficient and cost effective positive control. Do you think LiCl will suffice? Also, how do I use it on my cells (concentration, time of treatment...). Any advice will be appreciated.
Thank you
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