Hello everyone,
I am studying the expression of a cell-surface protein, on untreated vs treated cells. All of my cells even untreated are 100% positive when stained. So I do not have half positive and half negatives, it's all 100% positive. To analyse changes in all my 100% positives data, I'm taking the median fluo intensity of each condition, and I'm dividing each by the negative/isotypes values corresponding. Then I analyse the increase of that ratio between treated vs untreated conditions.
The thing is, I am not sure this is the right way.
Would it be better to substract the unmarked values to the marked ones ? or divide ?
And should I substract/divide all my data by one identical unmarked value ? Or should I continue to divide each marked value with its corresponding unmarked value ?
My unmarked median values are low but not constant (from 0.02 to 0.5 for instance in the same cell line), and my marked median values are increasing with the treatments. But when I divide each values by its corresponding unmarked values it can change a lot the results depending on the unmarked value... Is it normal and correct or does this technique create a bias in the analysis ?
Thank you in advance for your time, if anyone is doing the same kinf of analysis I would really appreciate to know more about their techniques.
Best
Hi Justine,
I suggest you to read the Flow Cytometry book written by Michael Ormerod, so that you are acquainted with flow cytometry terminology. Subtracting mean or median of unstained cells from stained cells is called MFI-Mean Fluorescence Intensity and yes it is a correct way of presenting cytometry results.
I suggest you check your staining solution, titration value of antibodies used for this analysis and see if your daily QC of the flow cytometry is passing all of the criteria for QC. A simple pH change in your staining solution can end up with crystallisations which will lead to 100% fluorescence in your measurements. Antibodies with expired use date will also cause excess fluorescence alike the untitrated high concentrations of antibodies. If the cytometry used has not passed the daily QC this will also cause false measurements.
Hope this brief information helps. I strongly suggest you to read Ormerod's book as a start, then look for Flow Cytometry issues of Methods in Molecular Biology and Current Protocols for Flow Cytometry.
Best wishes
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