This is my first time to try measure intracellular pH but it is confusing about protocol.

I have BCECF/AM (216254, CAS 117464-70-7, Sigma) and i understood protocol briefly as below.

1. Load BCECF/AM in cell for 30 minutes

2. Wash cell and eliminate extracellular BCECF/AM

3. Takes a fluorescence microscopic picture

or read fluorescence in microplate reader at both of 440 nm and 505 nm of excitation wavelength and at 535 nm of emission wavelength.

4. Calculates ratio of emission fluorescence (440/505 nm)

This pH measurement don't requires any standard pH solution to confirm exact intracellular pH?

How can i confirm that? or did i misunderstood protocol?

I want take microscopic pictures and measure fluorescence with microplate reader.

I also saw some paper that calculated fluorescence intensity from microscopic picture.

How can i calculate fluorescence with microscopic picture?

I'm so confusing now. Could you someone give me right direction to pH measurement?

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