If I want to compare MFI values across different experiments, is it important that I use exactly the same PMT voltages? Is slight differences be ok?

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Abhi Jayasimhan

No you cannot alter PMT voltages and compare MFI.

Md Babu Mia

Hello Mr. Sharon Lee ,

It's better not to change PMT voltage if you are intended to compare results. Your PMT voltage has significant role on MFI.

Regards,

Babu

Sharon Lee

What if I did not change the PMT voltage of the laser for the marker of interest, but changed the PMT voltage of the laser for identifying markers (e.g. CD8 to identify T cells or for DAPI to identify live or dead cells)?

Hello Sharon,

Ideally you shouldn't touch the PMT if you want to compare MFI. In your case if your population of interest is independent of your new marker (Altered PMT) then you need to check the compensations. Having said that it is not an ideal scenario to compare the MFIs after changing PMTV.

Thanks Abhi, could you elaborate on what you mean by "check the compensations"? Thanks..

Hi Sharon,

Lets say you want to compare MFI for FITC channel across experiments. Then you cannot alter PMTV for FITC. Now in your case if you alter PMTV for PE channel, then are you sure the compensation for FITC-PE remains the same ? What would the dim population in FITC channel look like? I think we should recheck the compensations if we have altered the PMTV. (flurophores with spectral overlap).

Experts in RG are welcome to correct me if I am wrong.

Thanks for your answer Abhi. Certainly the compensation would not be the same if the PMTV changes. But what if I had performed digital compensation at every experiment for the particular PMTV? My analysis for one experiment would therefore be based on the particular compensation matrix. Could I then compare the MFI values across experiments that had different PMTV (but were adjusted by the PMTV-specific compensation matrix)?

Could I then compare the MFI values across experiments that had different PMTV (but were adjusted by the PMTV-specific compensation matrix)???

I think you have phrased your question wrong. You cannot compare MFI for different PMTV even though you have compensated. Let me put it this way, if you have an unaltered PMTV for your marker of interest, and have an altered PMTVs for the rest of your markers (but compensated correctly) then you can compare MFIs for your unaltered PMTV across experiments. I hope you got it.