Hello,
I have been told that adjusting PMT voltages to lower compensation values is not a good practice. I found this PDF document (attached below) that is supposed to explain the impact of adjusting PMT voltages on spillover and compensation. I am not sure I understood everything, but I am still not convinced why it is a bad thing to do.
I am totally new to Flow Cytometry, any documents/tutorials/protocols that would help me with this technology (good practices, misconceptions etc...) are more than welcome!
Thank you,
Martin
Basically, compensation setups depends on your applications - example for immunophenotyping. Adjusting the PMT voltages should be done using the "unstained" cells and it must be done as follow :
The minimum PMT voltage setting for each detector is determined so that the resulting robust standard deviation (rSD) of the unstained cells is approximately 2.5x the robust standard deviation of the electronic noise (2.5 rSDen).
Purpose: To ensure that electronic noise does not interfere with measurements with dim fluorescence/negative events and that you are aware of these limits should it be necessary to decrease the voltages lower than these values.
Once you have got the unstained voltages correct, then run your individually stained cells - with optimized titrations.
Finally create the compensation and run a normal blood to verify your compensation setup. If there are issues such as spillover, check the information of the flourochrome/clones that you use.
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