I have isolated mitochondrial pellet from rat brain,
now how i can determine its calcium concentration using X-Rhod 1 ?
On surfing from net, the protocols are coming from cells.
But i have mitochondrial pellet from cells, so how i can proceed for calcium level in that pellet ?using X-Rhod 1 ?
Calcium indicators such as X-Rhod 1, Fluo-3 etc are commonly used to analyze the intra-cellular or cytosolic concentrations of calcium. For the purpose, cells are loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. The fluorescence signal from these cells is generally measured using fluorescence microscopy.
In few studies the calcium level is also measured in the cells as well as in the internal stores by using these indicators by monitoring the Ca2+ levels of the cytosol and of internal stores while manipulating nitric oxide. See the link:
A calcium-induced calcium influx factor, nitric oxide, modulates the refilling of calcium stores in astrocytes. doi: 10.1523/JNEUROSCI.23-32-10302.2003.. 2003 Nov 12;23(32):10302-10.J Neurosci
Article A Calcium-Induced Calcium Influx Factor, Nitric Oxide, Modul...
However, calcium indicator (AM Ester) like X-Rhod-1, AM uses intact cellular machinery to give the signal. The AM ester form of this rhodamine-based indicator is cationic, resulting in potential-driven uptake into mitochondria and a punctate staining pattern when loaded cells are viewed by fluorescence microscopy.
Because of this limitations, rhod-2 has been used in few studies as a selective indicator for mitochondrial Ca2+. However, the finding of localized subcellular accumulation on which the data interpretation is based is not universally supported.
Hence, either you go for the whole neuronal cells isolated from rat brain in order to measure calcium level using Fluo-3 AM or X-Rhod 1,AM or simultaneous measurement of cellular as well as mitochondrial calcium level indirectly using nitric oxide following the protocol given in the published paper above.
The third option is to transfer the mitochondria in lysis buffer, let the calcium come out (that still remain in the mitochondria) and then measure it Calcium Colorimetric Assay that is suitable for for use with cell and tissue culture supernatants, urine, plasma, serum, fecal material, media, and other biological fluids.
Calcium Colorimetric Assay.
https://www.sigmaaldrich.com/IN/en/product/sigma/mak022?gclid=Cj0KCQjw_4-SBhCgARIsAAlegrW5zHIOBAvAV41CHk8aJLvVSPyB8XAkqvSt37s630Dh0I2xz5dYAAwaAs_bEALw_wcB
Good luck
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