I'm working with primary human monocyte-derived macrophages. I had to suddenly switch from BD Accuri C6 to Facs Verse due to instrument brakedown, and I'm having trouble finding my macrophages in Verse fsc/ssc plots as the system has a very different working principle with adjustable pmt voltage. I tried to use BD-recommended voltages for fsc/ssc that work nicely for monocytes/granulocytes/lymphocytes according to frequent users of the intrument, but based on my previous data on Accuri I'm afraid that I'm not seeing the much larger macrophages at all, but only a small population of residual lymphocytes. Any advice in finding the correct voltage setup is much appreciated; could there even exist a BD-recommendation specific for macrophages?
I haven't used either of these machines, but do work with macrophages, they are indeed quite big and as you said you cannot use the same scatter pmt as for the lymphocytes whatever it is for the given machine. Set it to display max number of collected events to see everything well, then set both scatters to very low values (100? don't know how much it should be on this machine. basically until everything is squished almost to zero on both axis) and while collecting events, gradually and slowly increase scatters one by one until the main population is unfolded nicely and positioned on the plot the same way as you had it on Accuri. These are BMDMs so you should have a nice uniform population. My suggestion is that both voltages should be 1.5-2x lower than those used for the lymphocytes on that machine.
Thank you for the guidelines Oleksandr!
I seem to have adjusted the scatter voltages with too high increments at first & also both at once, which made it really hard to make any sense of what I was seeing. Now that I had some extra cells for running some more testing with the setup I managed to find my macrophage population. I new that these are big cells, but now that I saw the actual comparison in size to the blood immune cells - well, they are massive and require very different voltage setup.
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