I am trying to analyze Endothelial Microparticles in a FACSVerse, however I am struggling to set up the cytometer.
FYI: Threshold is 200 on FSC
PMT: FCS 186, SSC 250, FITC 430, APC 476 & PE 420
Please see file attached; top plot is mine and I need it to be similar to the bottom one.
Also I am concerned about the sizes: I don't understand why I am not getting the MPs in a smaller FSC.
If there is any "Flow Guru" out there, I would really appreciate its help!
Thanks very much
Hi Daniel,
looking at the plots I would lower the threshold on the FCS even further. At the bottom plot the threshold is somewhere between 0 en 10, while yours is at 200. Put your axis on bi-exponential display. If your microparticles are fluorescent in one of the indicated channels, it would be better to select on their fluorescence and backgate to a FSC/SSC plot, instead of only on their FSC/SSC properties.
Good luck!
Dear Bert,
Thanks for your answer!
I can not lower my threshold, as 200 is the minimum for the FSC in the FACSVerse and as far as I am concerned it should be enough (some people are acquiring the same MPs with a 500 threshold in the forward!)
I will try what you suggest and backgate the MPs from their channel instead of directly from the FSC/SSC,
Thank you very much for the suggestion!
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