Hi everybody!
Would be very thankful if someone can help with a small problem!
Suddenly HEK293 became insensitive to Carbachol stimulation in FURA experiments. With application of Carbachol no store-depletion is observed. Conditions are not changed, used the same buffers as before. Could it happen due to the lack of Muscarinic receptor? If anyone has ever had such problem?
Thank you for any kind of suggestion!
Hi Oleksandra.
Would you mind to give some more information?
What do you mean by "suddenly"? You tested the cells in Ca-Imaging one day and the other day no response was observed? Do other stimuli respond? Do your cells look "normal"?
Is your carbachol okay? How many times did you propaget these cells?
I do not have experience with Carbachol on HEK cells but I have experienced the same thing with ATP. Usually this kind of problem is caused by something trivial.
If you gave me more information, maybe I could help you.
Hi Timo,
I did not try other stimuli because I need exactly Carbachol response.
Yes, Carbachol is ok. Passage number is quite low.
When the cells stopped working, I thawed new one, but it did not help.
Then the only suggestion was that we had inappropriate FBS, because the company stopped producing it and we had to switch to another FBS. So now we checked different FBS but cells still do not respond to Carbachol.
I make the same buffers and split my cells in the same way as I always did.
Will be very thankful for any suggestion!
Hi Oleksandra,
We have had the same problem with carbachol on HEK cells, they suddenly stopped responding, althought we think we are doing exactly the same as before.
Please let us know if you have found a solution. Did changing the FBS work? Do you know if the muscarinic receptors are affected by trypsin? Do you know for how long carbachol is stable?
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