Hello Sanchaita Misra

In case of confocal imaging, you may measure intracellular calcium by using fluorescent indicator dyes that sense calcium. The confocal microscope detects the fluorescence of the dye, which is limited to a specific focal plane. This allows for precise measurements of calcium concentration and activity.

The protocol is simple. Just load calcium indicator dye into cells. With the help of confocal microscope, you may detect the fluorescence from the dye and analyze the fluorescence to determine calcium concentration and activity.

You may want to refer to the chapter attached below which describes sample preparation, fluorescent probe loading, labeling of intracellular compartments, and setting of parameters as well as protocols for measurements.

https://pubmed.ncbi.nlm.nih.gov/28116716/

As far as measuring intracellular calcium using FACS is concerned, you may use calcium-sensitive fluorescent dyes, such as Fluo-4 AM, indo-1, and fura-2. You may want to refer to the articles attached below for the protocol.

Article A flow-cytometric method for continuous measurement of intra...

Article Measurement of Intracellular Calcium Ions by Flow Cytometry

Best,

Malcolm Nobre Thank you for your response. If I fix the cells with methanol, is it still possible to measure intracellular calcium using confocal microscopy?

Hi Sanchaita Misra ,

I do not think so, since fixation will destroy the membrane integrity and Ca2+ will leak out of your cells.

Best Soenke