What is the best concentration of BAPTA?

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Carlo Manno

Hi Yu,

Everything depends on what you call abnormal increase of calcium. That is too little information.

I personally use 5 mM BAPTA (salt), using, of course, programs (i.e. maxchelator) to calculate the total calcium you need to use to have a constant [Ca2+]cyto, when voltage clamping skeletal muscle fibers. I generally don't like AMs because they go everywhere inside your cells (not only the cytosol). If AM is your only possible choice, I would look for methods with concentrations and "times" for this type of loading. Be aware that BAPTA-AM could be drastic lowering not only Ca but Mg as well.

Chenglong Yu

Hi Neetu,

Thank you for the suggestion. I will try with that concentration with cortical neurons. Maybe I can update the result later.

Hi Carlo,

That is a very good point. I do not like BAPTA for the same reason as they affect Mg. I am using GCaMP6f, and found that the Ca influx increased almost 1 fold for my neurons than control. I suggest to block some Ca channels but my advisor like BAPTA a lot. Do you have any hint about time when BAPTA-AM could cross a membrane? I mean the time window between entering the plasma membrane and before goes to others.@Carlo Manno

Thanks a lot.

Yu,

I haven't worked with BAPTA-AM however, esterases rapidly cleave the AM groups, so if you don't find a paper explaining in much detail this method, you'll have to try different times and concentrations by yourself (I would suggest no more than 15 minutes of loading at low concentration). I would alternatively suggest you to inject this cells with an internal solution with BAPTA (salt) and the fixed [Ca] and [Mg] you want to maintain constant (use the maxchelator for this purpose). However, I know this is not a fast/easy method.

Good luck!!

Thanks a lot for the suggestions. It sounds not easy at all for screening the best time and concentration.

Anyway, thanks for the suggestions.@Carlo Manno

Have a nice day!