recently I run a Portca assay 20 plex and I face a problem. The problem that I face is the MFI for my samples are very low (less than Zero). For standards the few first high dilution the MFI are good but suddenly in the low dilution MFI drop.
Could you please help me with the problem that happened? What are the possibilities that thing happend?
I think you used too low PMT voltages when doing flow cytometry. You have to set your voltages before acquiring on cytometer, for example using your background control. If you set your voltages to get a MFI with background of around 10-100, you should be able to get good standard curves.
Are you sure you are looking at raw MFI values? An empty Luminex bead usualy gives ~8MFI, Maybe you have set the system to report netto vallues, eg. (sample value) minus (control sample value). That could result in negative results.
The net MFI might be a possibility as Achterberg mentioned, though MFI shouldn't change with dilution. Other things to consider are proper calibration of the luminex plate reader (using non-expired beads), low bead counts, and use of appropriate diluents to dilute standard and samples. I guess another possibility is that not enough fluorescent streptavidin-fluorochrome was added.
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