I have analyzed platelet surface receptors using flow cytometry. The platelets were isolated from PRP, so, the samples contained only platelets. Every sample was stained with FITC-labeled antibody.The platelet population was identified and gated based on their characteristic forward (FSC) and side angle scatter (SSC) properties. The mean fluorescence intensity (MFI) was analyzed on a 4 decade log scale (1–10000); 10.000 cells were analyzed for each sample.
The reviewer pointed out that "data are not interpreted correctly and MFI vs percent positive cells should be checked. If there is a bimodal distribution of fluorescence it is inappriopriate to use MFI'".
What should I do? Is MFI, in this case, wrongly used? What is bimodal distribution?
Hi Alicja,
please see the attached link for a detailed explanation on when or when NOT to use MFI.
http://technical.sanguinebio.com/understanding-mfi-in-the-context-of-facs-data/
Without knowing the details of your experiment or your FACS plots, I would say that the reviewer is correct as you most likely have a non normally distributed dataset. My suggestion is to show (and statistically compare) the percentage of positive cells (meaning cells that stain with your antibodies vs. flourescence minus one (FMO) controls).
Good luck with your experiment & your reviews!
Michael
Hi Alicja,
please see the attached link for a detailed explanation on when or when NOT to use MFI.
http://technical.sanguinebio.com/understanding-mfi-in-the-context-of-facs-data/
Without knowing the details of your experiment or your FACS plots, I would say that the reviewer is correct as you most likely have a non normally distributed dataset. My suggestion is to show (and statistically compare) the percentage of positive cells (meaning cells that stain with your antibodies vs. flourescence minus one (FMO) controls).
Good luck with your experiment & your reviews!
Michael
Dear Michael,
Thank you for the help and very constructive feedback. I will take it into consideration.
Best wishes,
Alicja
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