We have a Beckman Coulter flowcytometer (2 Laser, 8 parameters). Gain of the FSC adn SSC is adjusted accordingly to separate two merging populations. There is option to select different predefined values from drop down menu in gain column (multiple values in FSC and SSC channel but only 2 values in fluorescence channels). For FSC we adjust gain upto "5" and for SSC we adjust gain from 5 to 50 on peripheral blood samples. but never played with the gain values of fluorescence channels.
My question is:
1. Are we allowed to play with the gain values while acquiring samples?
2. In what conditions should we adjust gain of fluorescence channels?
And any other comments/suggestions regarding adjustment of gain setting in fluorescence channels is welcomed.
Dear Jitendra,
I am not sure if I understood exactly what you mean by asking "Are we allowed to play with the gain values while acquiring samples?"
I am assuming a scenario where you are comparing Sample A vs sample B and these samples have been processed and stained using same protocol.
Now if you wish to use different gain values for sample A versus sample B in the same Flow cytometry run, then this would not be appropriate. You should use the same gain settings throughout for all samples so that they can be compared and analysed together.
I always use a isotype control and a positive control to determine the range of my gain settings before starting the final acquisition for all my samples.
However, it is of course possible to change the gain settings between different flow cytometry runs. If the cell types being acquired are different or they have been processed/stained differently, then they might need different FSC, SSC , gain settings.
Also, every device has its own dynamic range within which the gain values change linearly and are acceptable to use.
Best wishes
Shambhabi
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