Hello;
I've treated my cells with IFN-gamma to induce PDL-1 expression. Then, I investigate whether a synthetic peptide specific to PDL-1 could bind PDL-1, through flow cytometry.
To show peptide binding properties, I should report positive cell percentage and to show induction level, I should report MFI; Am I right about that? Is there any correlation between these two parameters?
I'm totally confused!
Any suggestion is appreciated :)
Percent positive is to separate those cells that has your peptide bound to it versus those that don't.
MFI of the positive cells can help you determine relative amount of peptide is bound to the marker on a per cell basis. So a higher MFI means the cell as a population has more peptide bound to the marker. For antibodies, it would mean that as a population, the cells have higher expression of the marker.
Since you are testing the binding of a peptide to a marker, just want to make sure that you also have antibody controls in there to determine the actual expression level of the marker. And also to do a competitive binding.
Hi dear Raheleh,
Yes, I think you are right. Positive percentage of the cells indicates the cells that express the desired marker and as your marker is a peptide able to bind PDL-1, therefore positive cells represent the cells that have been able to express PDL-1 following your treatment. The value: (Positive cells/total cell population* 100) could be regarded as a ratio of your induction efficiency assuming that your peptide has a 100% binding property and can bind any single PDL-1 expressed. Because you do not know how efficient your peptide binds the expressed PDL-1 and in other words binding potential of your peptide is unknown to you, it is recommended to apply an antibody specific to PDL-1 to show the real number of the positive cells (the cells able to express PDL-1 as a result of your IFN-gamma treatment) and compare this data with the data obtained following treating cells with your peptide in order to have a good estimation of peptide-binding affinity and property. But regarding MFI plots, we have two axises: one (for example X axis) representing mean flourescence intensity or MFI and another (the Y axis) shows the number of cells. In this plot on the X axis, as we go further MFI increases, also on the Y axis as we go further or upper the number of cells increases. As this plot is a bell-shaped plot (Gaussian plot or Gaussian distribution like normality distribution plots) if you consider any point on the X axis (a specific MFI) you can have an estimation of the number of the cells with that MFI and larger MFIs usually correspond to lower cell numbers as observed on the MFI plots. So, having MFI plot (MFI as X axis and cell number as Y axis) you can estimate how many of your positive cells have a specific MFI which corresponds with the cells with the marker. Cells with higher expression levels of PDL-1 on their surface have larger MFI values on the X axis and by observing the related value on the Y axis you can estimate the relative cell number having that MFI among total positive cells. So, you are right you can show induction level in the population of positive cells by reporting MFI.
Hope these can help you dear classmate.
Dear Mohsen Rastegari-Pouyani and Shen-An Hwang ;
Thanks for your explanations. Actually I don't have access to such antibody and I have to confirm both induction and binding through my peptide.
Hope to go through that successfully!
Is there no way to get any commercial antibody? Mostly because you will need some sort of control since your peptide is the variable. Without it... peer review will be tough.
Mohsen Rastegari-Pouyani has explained thoroughly and completely right. Thanks
old thread but see also Correlation Analysis of Intracellular and Secreted Cytokines via the Generalized Integrated Mean Fluorescence Intensity (GiMFI)a
Parisa Shooshtari,1,3,† Edgardo S. Fortuno, III,2,† Darren Blimkie,2 Miao Yu,1 Arvind Gupta,3 Tobias R. Kollmann,2,† and Ryan R. Brinkman1,4,†*
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