Hi Patrice,

In HUVECs 5 microM BAPTA-AM could not completely block the Ca-signal detected by Fluo-4-AM. We used 12,5 microM to get a complete inhibition. I have no experience with ruthenium red but in theory effective ion-channel blockers should work in much lower concentration than ion-chelators, since 1 channel delivers thousands of ions.

Ruthenium red will also inhibit SERCA so if part of your calcium response detected with Fluo-4 is ER calcium release (i.e., CICR), RR will reduce  the overall response.

Patrice, Hi!

With Ruthenium red an important issue is purity. If you use just the commercial RR, it will not work.  I have attached my bookj available on internet, which in the Supplements describes the prcedure for purifying RR and the reference.

Sincerely, Sasha Panov

Hi Patrice, 

I agree with most of the previous comments. It seems that you haven't use a high enough [BAPTA-AM]. However, when using an AM version, of both, the chelator and dye, they will enter any compartment with esterases (including SR and mitochondria). This will decreases enormously the dynamic range of your signal and will give you confusing results.  

My advice will be for you to inject both the chelator and dye in its salt version, you'll see a huge change. On the other hand, I've used Ru360 to block the uniporter, is a little bit unstable but if you work in the right conditions is more effective and specific than RR.    

Good luck and let us know your results. 

Carlo

Hi Patrice,

Concerning Ru360 applied to whole cells to inhibit the uniporter, I agree with Carlo that it might be better than RR, but in our experience, bath-applied Ru360 reduced Ca2+ influx via voltage dependent Ca2+ channels (in motor nerve terminals of lizards (1) and mice (2).  Perhaps you would need to also inject the Ru360 to achieve the "specific" effect of uniporter block.

All the best,

Gavriel

(1) David, 1999 (http://www.ncbi.nlm.nih.gov/pubmed/?term=10460256).

(2) Nguyen et al., 2009, supplemental Fig. 3 (http://www.ncbi.nlm.nih.gov/pubmed/?term=19174508).

As inhibitor of Ca transport works fine. The problem is that commercial RR MUST be purified. I did it/ an  it worked stably for many years.

The simple purification method is described in my book Practical Mitochondriology with original reference within. The book can be freely uploaded from my ReseachGate account/

Just as a guess, the Fluo-4 fluorescence decreases in RR because RR blocks Ca2+ channel/s on the plasma membrane (resulting in a decrease in the resting cytosolic [Ca2+] detected by the dye). You might check if a similar decrease in Fluo-4 fluorescence is produced by Ca2+-free extracellular solutions (buffered with ~ 2 mM EGTA or BAPTA, not AM). 

Thnaks for all thes eresponses which are really useful. We are taking most of the given  informations into account to drive more properly our experiments. Thanks