Hey guys,
I had this issue with my cells... I'm currently setting my experiments up, and the last time that I tried to stimulate my cells (through field stimulation) I couldn't get any calcium signal (even with 80V stimuli).
Despite the fact that the plate wasn't really confluent, neurons seemed fine (DIV14-18), and they were well loaded with fluo-4. Also, stimulator and software (HCImage) were fine. So, we think the problem is, in fact, those neurons.
Have you ever had this problem before? What was the cause?
Thanks a lot,
Fernanda
Are you sure that you load cells with Fluo-4, instead of Fluo-4/AM? Fluo-4 itself might not get into cell interior. In Fluo-4-loaded cell, simple method is that cells are bathed in calcium-free medium and then calcium (around 2 mM) was added to the bath and to observe if any changes in calcium transients occur.
Yes, I used Fluo-4-AM. I was using Tyrode's solution ([Ca2+]=1,8mM) as bath solution. I have also tested another Tyrode's solution and nothing happened. Anyway, I will try to put more calcium on the chamber today to see if I get any signal.
Usually, Fluo-4/AM needs to be added with cells for about 30 min. Alternatively, if you used Fluo-4/AM to load the cells, did you remember to wash out Fluo-4 from the medium? Are you sure that Fluo-4 was only contained inside the cell and bath medium should not have any Fluo-4 left. Since hippocamal neurons are excitable, it seems unlikely not to measure calcium transients.
Yes, after 1h of loading I washed the cells and left it for 30 more minutes in the incubator before I started the register. Probably, the isolation of the hippocampus wasn't good enough, so I didn't the proper cells. I will let you know if it was this problem. Thank you very much!
Dear Florian,
Thank you!!! I will keep that in mind, and see if that was the problem (i guess it was).
I'll let you know about any progress!
Hey guys, just to let you know, I have improved my culture and I found out that the problem with the stimulation was actually because the current was escaping from the chamber on the microscope. Hopefully, everything will work out now! Thank you for the valuable advices!
- Badges
- Science topic