I performed X-Rhod 1 staining for determining calcium concentration in rat brain mitochondrial fractions.

But, after calculations, in some samples the concentration is coming negative, which is ofcourse not right. I want to know the reason behind that ?

Rmax (flourescence intensity of calcium saturated sample) was coming less than R(flourescence intensity of my sample)

As far as i know, concentration cann't come in negative value.

But, after calculations, in some samples the concentration is coming negative, which is of course not right. I want to know the reason behind that ?

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