I am using MCEC (Mouse cardiac Endothelial cell line) and using Methylglyoxal as agonist. I would like to know what could be possible ways to explain this phenomenon. I have also tested and it is not an artifact as agonist is not interacting with FURA dye.
Hi Robin,
It is hard to tell without knowing which agonist you are using...
Dear Norbert....
Thanks for your response
I updated my question. I am using methylglyoxal as agonist on MCEC (Mouse cardiac Endothelial) cell line.
I believe you apply methylglyoxal with a perfusion system - have you excluded any effect of the perfusion itself - i.e. do you have a constant perfusion of the control solution between agonist applications?
Also keep in mind that methylglyoxal is a very "dirty" molecule in a way it has many unspecific targets. This being said, if methylglyoxal does something on you intracellular levels you would rather expect an increase of calcium as methylglyoxal is known to activate some plasma membrane channels including TRPA1, but also may trigger calcium release from internal stores...
In your case you rather observe a decrease of intracellular calcium levels. What I could imagine is that your cells have a passive calcium influx that may be blocked by methylglyoxal. Not sure it makes totally sense but what you may want to do is to repeat your recording in the absence of extracellular calcium and see if you still observe the same effect or not.
In addition to Norbert's helpful suggestions... this can happen if compounds affect Fura2 directly. Caffeine has such an effect (Biochemical Journal 329, 349-57). One way of checking this is to deplete all intracellular calcium stores (e.g. with application of ionomycin in calcium-free medium) to prevent any calcium flux in response to your agonist. Following that treatment, if your agonist still changes the 340/380 ratio then it could be a direct effect on the indicator. Can you switch to a different calcium indicator (e.g. Fluo-8, or a ratiometric pair of Fluo-8 and FuraRed)? That might alleviate the issue.
Can you show us the 340 nm and 380 nm traces rather than just the ratio? If it's a genuine calcium change there should be corresponding reciprocal changes in the 340 and 380 traces.
Best wishes,
Martin
- Badges
- Science method