Hi Aurore, what is the initial ratio? Was that F340/380? The baseline seems short compare to other curves. It will be helpful to find out the problem if you have slightly longer baseline. What's the reason to incubate cells with calcium? What kind of cells was that?

You need to ensure that you used Fura-2/AM instead of Fura-2.

Hi Aurore,

A possibility is that you have some Fura2 leaking from your cells. Many cell types are good at extruding calcium indicators, particularly at 37oC via ABC transporters. If Fura2 is exported from a cell it will encounter a saturating calcium concentration (even in your Ca2+-free HBSS medium), and will therefore give a high starting 340/380 ratio. Since you are using a spectrofluorimeter (with cells in a cuvette?) the extruded Fura2 will generate a signal from a high calcium environment.

Some of the fura2 is obviously inside your cells, since they respond to ATP. The addition of EGTA (presumably in large excess over calcium) at the end of the experiment will greatly reduce the free calcium concentraion in the medium outside the cells, so that there is (i) no longer a signal from the Fura2 within the cells, and (ii) the Fura2 outside the cells is no longer saturated.

You can inhibit extrusion of calcium indicators using compounds such a probenecid and sulfinpyrazone. Alternatively, if you do experiments at 20oC extrusion of indicators is much less of an issue. We tried to give some tips about preventing extrusion of caclium indicators in this article: Article Loading Fluorescent Ca2+ Indicators into Living Cells

Another way of tackling this issue is to do single cell imaging and to perfuse your cells with ATP, CaCl2 and EGTA, in whcih case any extruded Fura2 would be washed away.

Extrusion of Fura2 could explain a high starting 340/380 ratio and why EGTA gives a lower final basal ratio, but doesn't really explain why there is a large drop in ratio after ATP addition: that is a puzzle. Did you add a large volume of ATP, so that your cells were diluted in the cuvette?

Is there bleaching of the Fura2? This would be evident as a time-dependent decline in the fluorescence of both 340 and 380 nm traces. The 340/380 ratio is quite resistant to bleaching, but sometimes not fully. You could try to reduce the 340 and 380 excitation light intensities.

Best of success with your experiments.

Martin

Dear Aurore,

Loading at 37°C sometimes is... tricky :) I had bad experience with fura-2 going to internal stores.... as the Kd of fura-2 for Ca2+ ions is around 250 nM, in the ER, fura-2 is saturated... it could explain why you have a high baseline.As your cells respond to stimulation, it means that the majority of your fura-2 is in the cytosol.

I use to load epithelial cells at room temperature under slow shaking.

Sometimes, and especially if you use primary cells, they are more fragile than cell lines. You can try to load them without extracellular Ca2+ ions for 30-45 min. Then store them still without Ca2+. And add Ca2+ when you start the experiment, you will see an increase due to Ca2+ leaking in the cells, and after 1-2 min the [Ca2+]cyt should be stable, and perfect for your exp :)

Best success in you exp :)

Olivier

In addition to the leakage of fluorophores, I always had troubles to measure ca2 + in cells that express the purinergic receptor p2x7. When I was an undergraduate trainee, I published a paper about the problems of use fura-2 and ATP in some cells. Doi 10.1023/B:JOFL.0000047221.51493.e3

Paredes-Gamero EJ, França JP, Moraes AA, Aguilar MO, Oshiro ME, Ferreira AT. Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2. J Fluoresc. 2004 14(6):711-22.

Best of success with your experiments.

Edgar

Edgar, I agree with you :)

I use to work on cell lines of B lymphocyte type, and some cell lines "like" Fura-2, some others "prefer" Indo-1...

This resemble very much indeed a P2X7 mediated effect.