Here's a tough one that I can't figure out..... A colleague of mine transiently transfected HEK-293 cells with message for CMV-driven yellow (Topaz) fluorescence protein. He brought the cells up for me to analyze on our cytometer. I used 488 excitation and read out both FL1 and FL2 log signals. The gains were set as usual, so that the sham-transfected control (auto)fluorescence was set within the first decade. For the Topaz transfected cells, I observed about 40% of the cells fluorescing under the microscope. But when I analyzed them on the cytometer, EVERYTHING was shifted to the right, including the negative subpopulation of the transfected sample. The ~%40 percent positive subset was clearly discernible from the "right-shifted" negative population. Of course this suggested that there was an autofluorescence issue, but this was clearly not observed on the microscope using similar filter arrangements. A couple of other issues for you to consider with this experiment: 1- the "positive" cells were incredibly bright on the microscope 2- when we mixed the sham-transfected with the test cells, the negative peak of the sham-transfected cells moved right as well!! What is going on here? Is there such a thing as a "lantern" effect of very bright cells in close proximately with negatives? Is fluorescent subcellular debris confounding my light scatter gating? Please give me your explanations because I've not seen this phenomenon before in flow. Phil Marder Lilly Research Labs _________________________________________________________________ Get your FREE download of MSN Explorer at http://explorer.msn.com/intl.asp
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:58 EST