[Cytometry] Problem with fluorescence signal reduction
F.Tommasino at gsi.de
Thu Apr 18 10:22:26 EDT 2013
I would like to ask for your opinion concerning a problem I'm recently experiencing with my flow cytometry measurements. I use to do intracellular staining on fixed cells for a DNA damage marker, which consists of double antibody staining. The signal is then measured in the FL1 channel of the Partec PAS III available at our institute (excited by a 488nm laser). Since I need to compare measurements in a quantitative way, I use to add beads to my sample in order to have a "real-time" reference.
Since some weeks I have observed a reduction in the order of 30% of the signal coming from the cells, but not of the signal coming from beads, that stays stable. Obviously, I thought about problem arising during the sample preparation (PFA 2%, Triton X-100 0.5%, BSA 0.4% are used for fixation and permeabilization). I tried to prepare fresh solutions, as well as I tried with different antibody stocks, but still I observe a reduction of the signal compared to previous measurements, also using different cell lines.
I'm also carefully checking the machine, I did repeated cleaning cycles, and finally I replaced the Flow Cuvette with a new one, having in mind that eventually some alteration in the fluidic stability of the system could affect cells but not beads which are smaller.
It would be nice if I could get any feedback concerning what are the aspects which I still could check, or which test I could repeat.
I thank you all in advance.
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