I have performed a antibody titration to detect a specific cytoplasmatic protein in human monocytes starting with the double amount of general antibody recommendation (2 ug) and I have made 9 dilutions with the half of the antibody concentration of the previous dilution. Most people recommend stain index (SI) to determine the best amount of antibody, that is determined by the difference between mean fluorescence intesity (MFI) in positive population and MFI of negative population divided by 2x standard deviation (SD) of negative population, however, I have notice that data regarding the events in my assay are non-parametric and use mean is not aplicable and SD is higher than mean. I have found another way to performe stain index that consider median fluorescence intensity rather the mean in this formula, that I believe is more aplicable in my case. So, SI = difference between median of fluorescence intensity in positive and in negative population divided by the difference between 84% of negative population median fluorescence intensity x 0,995. Someone recommend this formula to determine the best amount to antibody to my future assays?
Someone can explain me what means 84% of positive population referenced in this formula? 84% of median fluorescence intensity?
I attached the formula and my results with different antibody amount in human monocytes.
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