Could any one say how to detect calcium concentration or calcium flux out of cell but not within the cell? Please also help me for calcium indicator for the same.
Hi Ramiz,
To look calcium fluxes, the best choice is measuring ion currents through calcium permeable ion channels.
Using for this purpose any calcium dye I believe would be impossible because volume of the cells is much more less than the volume of the bath, even if you make very small chamber. It will be very difficult to avoid a huge error. Also extracellular calcium is present at very high concentration compared to Kd of calcium indicator. Fast search give me some article where people try to measure extracellular calcium.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441573/pdf/nihms52310.pdf
https://aip.scitation.org/doi/pdf/10.1063/1.5038489
Good luck.
Hi Ramiz,
I'm not fully sure what you're trying to measure, but if you want to follow the dynamic flux of calcium out of the cell you could use the method developed by Alexei Tepikin and colleagues. They used a combination of fluorescent calcium indicators to simultaneously measure calcium inside and outside of single cells:
J Biol Chem. 1992 Jul 15;267(20):14073-6.
Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking.
Alternatively, calcium flux from the cell can be monitored by using radioactive calcium (45Ca2+). There are published methods for this.
I hope this helps. Good luck with your experiments.
Martin
Hi Ramiz,
The quantification method for detecting intracellular and extracellular calcium concentration proposed by the Nobel laureate R.Y. Tsien in 1980 (Article New Calcium Indicators and Buffers with High Selectivity aga...
). And it is helpful for accurate determination of the intracellular and extracellular calcium concentrations using ratiometric fluorescent calcium dyesArticle Theoretical analysis on ratiometric fluorescent indicators c...
.
Which do you want? The molar value of extracellular calcium, or the moles of calcium leaving the cell? As the concn difference across the plasmalemma is 1000 fold, depending on the cell type and it's function, direct measurement/visualisation of efflux cannot currently be done as far as I am aware.
So estimate the no: of efflux channels/exchangers (radioligands) and then measure the flux (electrophys) and hey presto, 3-10 years later........
If you want the extracellular Ca2 concn either control the amount or use a pCa2+ metre.
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