Hi there,

It is hard to know what exactly is going on but perhaps we can do some troubleshooting here.

So from what I understand, you stimulate the fluo/rhode-loaded heart slices to contract, which they do, but even though they contract you do not see a calcium response?

(1) Are you sure the calcium indicator has actually loaded into the slices i.e. can you detect the fluorescence of your indicator in the absence of stimulation? With slices, you might need to incubate for a longer time to ensure successful loading.

(2) Have you tried eliciting a calcium response in different ways? Besides contraction, you could trying stimulating your slices with different agonists e.g. ATP that should elicit a calcium response.

I hope this helps at least a little bit!

Thanks John for your response.

1. When I used the fluo-4 there was some autofluorescence coming from the tissue, so it was hard to say if it was loaded with the dye or not.

With the rhod-2 I didn't have that problem and I saw a nice fluorescence signal coming out of it.

2. Can you tell me why do you think different stimulation approach will give different results. I'm not familiar with the of stimulation.

Thanks a lot

Hemi

Hemi Rotenberg

Hi again,

The idea of using various stimulation methods is to ensure your experimental set up is correct. If you can elicit a calcium response, say following ATP perfusion over the slices, this means the software is working correctly and that the tissue is indeed healthy & responsive.

As an aside, what software are you using to do your live cell calcium imaging? I still am a bit confused by what is going on. Do you initiate contractions and just see a flat "line" as a response (indicating no calcium response). Also does your lab do live cell calcium imaging frequently on the cardiac tissue slices?