Hi everybody, I have been trying to perform DNA/RNA simultaneous analysis using Hoechst and Pyronin Y to separate the G0/G1 compartments of ethanol-fixed cell lines that have been subjected to varying degrees of starvation. I’m seeing a strange drift in the fluorescence as the sample runs, and wonder if anyone else has seen something similar or has any suggestions. I have a FACStar+ equipped with an Enterprise laser. I’m using the 488 line to excite the Pyronin, collecting in FL2. I’m regulating on UV at 25mW, which puts the 488 around 110 mW (fairly stable) once the laser has warmed up. This provides good signal above background with linear amplification and reasonable CVs on the DNA histogram. What I am noticing: as the sample runs, the Pyronin signal gradually increases while the Hoechst decreases. The change in the PyY is more pronounced than the Hoechst, but the DNA histogram also moves up and down from sample to sample. Plotting PyY vs. time allows the delay of acquisition until the signal has plateaued (around 1-2 minutes). Then adjusting the Hoechst PMT to set the DNA histogram in channel 200 makes the data look nice, but its validity still concerns me (not to mention that this is extremely time consuming). Has anyone else noted this sort of phenomenon? Could some kind of fluorescence energy transfer be going on? If so, why does it take so much time? I would appreciate any tips on instrument set up or staining protocols that may alleviate this, and input on how it may be influencing the data. Thanks, Cathy Allen VA Medical Center Nashville, TN.
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