We have observed a decreasing median fluorescence intensity (MFI), and recently extracted data from seven years of analyses (se attached figure) showing a very clear decline in fluorescence intensity. We do not think this has to do with reagents or antibodies. Could it be the red laser declining in activity? Could we somehow compensate (eg. by increasing voltage in the flow cytometry protocol)?
Background: we are running platelets and detect a panel of platelet surface glycoproteins using labelled antibodies. We use a Navios from Beckman-Coulter
Any suggetsions is appreciated.
This happens with every instrument based on laser, as lasers have their defined life (few thousand hours). So, at voltage "X", you get MFI "999" as the time ( consumption hour) passes, your laser weakens so you give "X-x" voltage, so it leads to decreased MFI.
With time, you need to increase voltages to make a constant incident light, which further will maintain the constant MFI.
There are certain beads (Flosser beads), which maintain the system for you. Once you run the beads and set the voltage, it set it for you.
Anything you find any changes in MFI (suspect of power of incident light), u need to run Flowset beads (according to manufacture protocol) to automatically set voltage to gain similar output.
Hope, it was helpful.
Peter,
You are right when considering the laser as a factor for the changes observed in the MFI. Laser power reduces with time affecting your MFI. Therefore, changing the laser between experiments that you would like to compare the MFI among samples is not a good idea. Other factors that can affect the MFI are the machine calibration and differences in compensation. I hope it helps.
Another contributing factor could be the alignment of the optics (mirrors and filters) and also laser delay settings for the instrument. Having a field service engineer look at these things might be a good idea.
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