![]() Gatings should not deviate much most of the time, however, clear positive and negative populations account for adjusting the gates due to sample-to-sample variation.ġ. We have had annual maintenance services from BD and the technicians have numerously said to us that even road construction outside our building might influence the machine (our lab is not a controlled environment as in the clinics).ĥ. Someone can literally just touch the machine and the compensation/lasers can change – it is impossible to know, so this is why the compensation has to be checked every time for every sample. Apparently, the instrument is very sensitive to external vibrations or even the countertop that it is standing on. The PMT voltages cannot be changed of course, but the compensation matrix has to be checked.Ĥ. I have been taught that this is because there can be huge differences even if the samples have been acquired with the same template and values. The analysis itself is done in batches but all the compensations are always double-checked beforehand. Thus, tweaking the compensation values is done carefully in order to adjust these discrepancies.ģ. Some samples have a lot of autofluorescence when compared to others. Some compensations look heavily under-/over-compensated. The matrix does not look alike for each sample. Because the compensation depends on the day and the sensitivity of the instrument varies throughout the years of collecting the data, this is a lengthy but crucial step.Ģ. Check the compensation matrix for each sample. This is what I have been taught thus far:ġ. I understand the concept of making correct compensations for the instrument itself, but I am now wondering whether I have been taught wrong about manually adjusting the compensation values in the compensation matrix when analyzing the data in FlowJo. Our lab is currently using BD FACS Verse (8 colors) to immune phenotype primary renal cell carcinoma tumor cells over the span of 5 years. Hi all, I’m a mere PhD student questioning my very own existence at the moment and in desperate need of some expert advice regarding flow compensations as well as the of tweaking of values in FlowJo.
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