I used two BD FACS machine. In FACS Calibur,I adjust the FSC-H Amp Gain. In FACS diva, I adjust the FACS scaling to make the same value of FACS-A and FACS-H. I wonder to know what is their principle and their difference. Thank you very much!
Someone can correct me if I'm wrong, but I believe there are two main ways to adjust the signal detected by the PMT. One is by adjusting the FSC voltage (which directly alters FSC-H, or the height of the signal) or by adjusting the amp gain.
Increasing the voltage will increase the magnitude of the FSC signal. Adjusting amp gain will alter the entire signal waveform by a factor of 1-9.99. The FACS Calibur software limits you on the voltage setting in a log 10 scale (up to 1000), whereas FACSDiva allows you fine adjust the FSC voltage (1-1000). This is why the amp gain is there on the Calibur, to fine adjust the FSC signal.
In addition, if you want an amp gain of 10, you simply need to increase the voltage by a factor of 10. I believe the main difference is that altering amp gain changes the entire signal waveform whereas altering the voltage affects the peak magnitude of the signal. Since FSC-A (area of the signal) is based on both FSC-H (affected by voltage) and FSC-W (width of signal or time, not affected by voltage), FSC-A can vary between "similar" adjustments with voltage and amp gain.
I agree with Stefanie to a degree that as long as all your cells remain on the plot you should be fine. However, you could alter results depending how you draw your initial gates. By results I'm assuming you mean the calculated frequencies and total number of CD4 T cells? These results will be based upon how you draw your previous gates, which may be impacted by how nice your populations stay consistent and segregated.
So if your first gate was on lymphocytes, but the way you draw your gate changes from experiment to experiment (due to changing the FSC/SSC), you could alter your results. Thus, you have to be careful how you want to analyze your data. The key is to keep as consistent as possible from experiment to experiment.
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