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Flow cytometry and immunofluorescent staining: help needed

histogram immunofluorescence percentage positive publication adherent cell

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#1 Mattjim

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Posted 05 February 2014 - 03:51 AM

Hi,

 

I'm involved in a paper where I have done some flow cytometry data collection and analysis. As requested by the first author I have done these plots in a histogram format and have shown percentage of positive cells.

 

The issue is that the author has combined the flow data with immunofluorescent data in a figure and was trying to compare her immuno against the flow. This is causing a problem I don't know how to explain correctly for publication purposes. Specifically her cells show weak staining over the whole population, whereas the 'percentage positive' is only 3%.

 

Looking on the histogram the whole graph has shifted up, so it looks like the whole population is brighter compared to the isotype, which correlates with weak staining.  

 

In short, how do I present/discuss this? Do I omit or ignore the percentage positive or is there a work around/sufficiently adequate explanation for publication?

 

Please help!

 

Thanks



#2 science noob

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Posted 05 February 2014 - 05:58 AM

Another crucial data which you can gather from your flow readouts is the mean fluorescence index (MFI) or simply the mean of the fluorescence staining.  This will take into consideration "weak" staining and not just % positivity.

 

Here are some good reads regarding MFI:

http://technical.san...t-of-facs-data/

http://ucflow.blogsp...hat-is-mfi.html

http://www.ncbi.nlm....les/PMC2822558/



#3 Mattjim

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Posted 05 February 2014 - 06:28 AM

Excellent, thanks!







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