Odd shift in FL2 values (newbie flow question) - (Dec/10/2007 )

Hi all,

I have recently came into posession of an older model two color BD FACScan cytometer. It is still in operational condition and I have been using it regularly for single color cytometry using FL1 (green). I have went through the process of setting up the instrument on my negative cells, and have adjusted the machine so that the cell population is clearly defined in the FSC/SSC density plot window and I have a nice negative ball of cells by the FL1/FL2 density plot. The problem is when I treat my cells with an Alexa488 labeled oligonucleotide the FL2 signal (red) gets floored (i.e., the entire population of cells goes from centering just below 10^1 to being flattened at 0). I don't know if this is due to the compensation settings or maybe an alteration of SSC caused by the dye. If anyone has had experience with this I would really appreciate input.

Thanks a lot!

would it be possible to show your plots?
What software are you using and are you using the exact same voltages as in your negative control cells?
Are your control cells treated exactly the same except for the oligo labeled with Alexa488?
What is your exact treatment actually? Permeabilisation/fixation etc?

Ok here are two example files. These are both RPMI8226 human myeloma cell lines that were cultured in identical conditions for 48h. The control oligo file shows cells that were transfected with a non-labeled siRNA oligo using HiPerfect transfection reagent, washed 3x in PBS+BSA, resuspended in 400ul of PBS+BSA and analyzed by flow. The cells in the positive oligo were treated identically with the exception of having a labeled siRNA oligo (Alexa488). Results are identical with paraformaldahyde fixed cells. The voltages, amps, and compensations were all set on the control oligo treated cells. I heard that compensations aren't usually set for single color analysis, but the cells were slightly autofluorescent and had a tail coming off of the main population of gated cells (lower left quadrant to upper right quadrant). Analysis was carried out on an older version of cell quest circa early 1990s.

Thanks for taking a look at this!