Dear Krishna,

It would be great if you could give some details of what you are doing so that it is possible to help you more specifically.

Fluo-4 is a calcium sensitive fluorescent dye (actually, it is not the best) and these dyes are very useful to monitor the intracellular free calcium transients during repetitive contractions of the cardiomyocytes and also the propagation of the calcium waves in monolayers of cardiomyocytes.

Which kind of cardiomyocytes (primary or stem cell derived) are you using and why do you need to centrifuge them?

What is the intended application you are looking for? Is it single cell video monitoring in a microscopic system (e.g. with the IonOptics or Sony System) or a kinetic plate reader assay system (e.g. Hamamatsu FDSS or Molecular Devices FLIPR)?

All the best,

Ralf

There is no doubt that centifugation stresses cardiocmyocytes particularly freshly isolated cells. I assume you are removing the Fluo-4 buffer after loading. Large swings in global Ca2+ impact on cell survival. Limiting centrifugation to very low rpms and duration can help. Aviod a tight cell pellet and even try a gravity filtration approach.

Good luck

Stephen

Hi Krishna,

Depending on your experiment you don't need to centrifuge, after loading you could place a drop of cells in your recording chamber and perfuse the chamber with fresh solution to wash away the extracellular dye.  Let the cells rest in the fresh solution for ~10 min to allow the dye to deesterify then you are ready to experiment.  Alternatively if you load the cells with dye in a vertical Eppendorff or test tube they will tend to sediment into a pellet then you can just remove the supernatant without needing to centrifuge. Bear in mind Fluo will buffer Ca2+ to an extent and reduce contraction.

Best,

Ewan

Dear Krishna, in my lab we just let the cells sediment. We load them with fluo-4 AM for 15 minutes in an eppendorf tube laying on the side, then for 5 minutes I let it stand and the cells sediment. I remove the supernatant, replace it with storage buffer, gently mix them and let the cells lay in the eppendorf tube on side. After another 15 minutes I again put the tube into stand, sediment for 5 minutes and replace the supernatant, after that I keep the cells in the tube laying on side and just take into the experiment as many cells as I need. If the isolation is good, the cells survive nicely and have good calcium signals.

Not necessary. After 20 min loading ,cells are already in the bottom pellet. Gently remove the supernatant and use fresh working solution to re suspend the cells.

Dear Dr.Ralf,

Thanks for your response.

I am using Fluo4 AM to image calcium transíents over a period of time in primary rat cardiomyocytes. I am looking for calcium transient variations within the excitable and nonexcitable cells together in the culture. When i looked up for a protocol, it suggested to centrifuge before the experiments. However, i understand that for my application it is not possible to centrifuge because i wish to image the calcium transients immediately. 

Regards

Krishna

Dear Dr.Bird,

Thanks for your response. 

Yes i agree. I feel that instead of stressing the cells, it is better for me to simply image after washing the Fluo4AM from cells. But, the two points you stated to avoid tight pellet and gravity filtration are helpful suggestions.

Thanks and regards,

Krishna

 Dear Dr.Fowler,

Thanks for your suggestions. I do agree with you that Fluo4AM affects the contraction over time.

 Dear Dr.Alexandra,

Thanks for responding. I really like your idea of letting the cells sediment instead of centrifugation. 

Regards,

Krishna

 Dear Dr.Zhao,

Thanks for responding. I will try it.

Regards

Krishna