Hi everyone,
I am trying to measure calcium sparks and transients in freshly isolated rat mesenteric smooth muscle cells using Fluo-4 (5 microM). we have a Zeiss LSM 900 confocal microscope that we use to image the cells.
Unfortunately, cells don't show any change in florescence after the administration of drugs. We have tried Angiotensin-II, caffeine, Calcium Ionophore (A23187) with no luck.
Has anyone faced any similar issue like this one? Does it mean the Fluo-4 has gone bad? I dissolve Fluo-4 with DMSO right before use. Fluo-4 loading is fine, since i can see the cells in AF-488 light.
Any help will be deeply appreciated.
Hi Sayeman,
Try stimulating with high potasium solution (ej 30 mM) they must depolarize and show a fluorescence response otherwise your system is the problem.
Best ragrds
Enrique
If you are not sure about the dye, you can try changing the buffer solution from normal to high calcium during the recordings and see if the signal increases
Hi Sayeman, may I know how have you solved this problem? I am facing the same issue in my experiments using HEPG2 cells. There's no change in fluorescence after adding KCl and ionomycin.
Hi Sayeman Islam Niloy , I'm trying calcium imaging with fluo-4 AM, in acute brain slices, but facing similar issues as you have mentioned, no change observed with drugs. Also, I'm not able to visualise the stained cells under the eyepiece of microscope in lower magnification, say 20x. Could you please help?
Hi Sumela Basu , my Fluo-4 went bad, that's why it wasn't working. I bought some Cal-520am and its working really well.
In your case I would suggest checking if the fluo-4 still works or not. If you have some HEK293 or any other type of cultured cells, I would load them with Fluo-4 and use Ionomycin to see the response.
If it's showing bright calcium signal I would focus on optimizing the loading concentration and procedure for you acute brain slices.
Good luck!
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