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Drift of fluorescence signal


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#1 Baloo

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Posted 30 July 2012 - 11:02 PM

Hello to everyone,

I would like to ask your opinion concerning a problem we have been experiencing during the last weeks with our flow cytometer. The problem is that when you start a measurement session, and you measure some beads, you get some MFI, but if you measure the same beads over time, what you observe is a drift of the MFI value toward lower values. Sooner or later this drift reaches a stable value, but it could need even several hours.

It's obviously not a problem of warm-up time, and it seems not being related to laser source instabilities, since the drift affects all the signals, also the one coming from the UV lamp.

Do you have any ideas or suggestions concerning what to check to possibly solve the problem?

Thank you in advance!

#2 Curtis

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Posted 31 July 2012 - 10:08 PM

I used to stain my apoptotic cells with PI. If I left the cells in PI for long time, even my controls/healthy cells would take up the stain. This is normal. you just need to find the best time to measure.

Do you keep your samples in dark?

Also, when I did immunofluoresence and observed my DAPI stained cells under microscope, just immediately after switching on the microscope, the DAPI of the area that I was looking at turned green, and after a while they looked less bright as they were before. DAPI must be blue, so to get better pictures I had to move to a new area and immediately take a picture. Otherwise DAPI would get burned out. I am not sure what the wavelength of the filter that I used was. Maybe it was UV? but whatever the wavelength it burned my DAPI-stained cells. What is your label?

#3 Baloo

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Posted 01 August 2012 - 06:56 AM

Hi Curtis,

thanks for your answer. Actually I also use to stain with DAPI for cell cycle measurements and to measure the signal with a UV lamp. The point is slightly different. I see this constant drift of the signal even when I only measured beads, which are stable for definition.

I think there is a problem hidden somewhere in the machine, but I don't know how to find it, and so I'm asking around...hopefully (for me!) someone already experienced this nasty problem!

#4 Curtis

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Posted 01 August 2012 - 08:55 PM

what beads are they?




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