Hello all,
I'm trying to establish a calibration curve and use it to find the intra-cellular pH of HGT-1 cells when treated with coffee samples. Overall, I use SNARF-AM and flow cytometry to measure the intra-cellular pH. Potassium buffer at different pH and median fluorescence intensity (MFI) data is used for establishing the calibration curve. What I've obtained from the calibration curve is a formula with negative slope: y=-0.1541x + 2.2039, R2 = 0.9737. When I key in my MFI values into the formula to find the x value, I've ended up with negative values.
So my question is, should I ignore the negative signs ? Because it is not possible to have intra-cellular pH to be negative. Can anyone help me to clarify this ? Thank you in advance.
You should not ignore the negative sign.
If your standard curve has a negative slope, it means that the measured fluorescence value decreased as the pH increased.
If you expected to get a positive slope of the standard curve, then you should examine the quality of your data to see what went wrong.
You can share the raw data if you want more detailed advice.
Adam B Shapiro Thank you for the advice.
Here I've attached the raw data that I have analyzed. I've key it into Excel.
The ratio value is final and subtracted with the background MFI.
It is concerning that you have such a high proportion of outliers. Further effort is warranted to collect a high-quality dataset.
Aside from that, the standard curve of the fluorescence intensity ratio FL2/FL3 versus the pH shows a definite negative slope. You should be able to use such a standard curve to calculate pH if the data quality is high. Do not attempt to extrapolate for F2/F3 values outside of the range of the standard curve, however, because the linearity may not continue outside of its boundaries.
Adam B Shapiro Thank you for the feedback, and yes I will need another attempt for high quality data of flow cytometry.
Let say with another attempt of the experiment and obtaining a good data with negative slope. Should the pHi still needs to be pH -7 ? Or I should just ignore the negative sign and reported pH 7 for coffee sample BC (black coffee) for example. Would that be recommended ?
Your standard curve looks to be useful between pH 6 and 9. The average of the 3 black coffee measurements was 1.151. Using the linear regression equation of your standard curve, which is y = 2.2501-0.161x (where x is pH and y is FL2/FL3), gives a pH value of (1.151-2.2501)/(-0.161) = 6.8. Since this falls within the range of the standard curve, it is a valid measurement.
I think you were getting negative values because you forgot to put in a (-) sign in the calculation.
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