Are you setting baseline voltages according to your unstained cells each experiment?.I'm not familiar with Verse (can you set voltages yourself?), so some of my questions may not apply: Lots of red-flags from your description, but there's still a lot more info needed. I would appreciate any insight on this matter! I’m so sorry for the rant and thank you to whoever is reading this drama. And since I’m not in the states, we haven’t heard from them in quite a while probably due to the midsummer vacations coming up. We have recently discussed this matter with some flow specialists from BD, but they really didn’t provide a clear-cut answer (it was more like: “the rule of thumb is not to manipulate the data, but carefully tweaking is a-ok”). Classic imposter syndrome sweats right here.Īnyways, despite trying to read up on this topic, I was surprised at how little info I could find and had to some digging on the web. I guess I did not question things that were established for more than a decade in our lab. And worst of all, I couldn’t really argue much with my other colleagues because it made me realize how little I actually know about compensations and flow cytometry in general. I am horrified at the thought that I have been doing my analysis incorrectly for the past four years. Even though there are sample-to-sample differences, gates should stay the same for all samples every time and should only be changed when absolutely necessary. Even though there are over-/under-compensated values in the matrix, it doesn’t really matter in the analysis, as long as the same compensation is used across all the samples.Ĥ. The acquisition defined matrix stands as it is in FlowJo, since the compensations have already been done from Verse – do not copy, do not edit, do not touch, do not do anything to it.ģ. Manipulating the matrix at any cost is forbidden, since the overall biology is then not accurately captured, and the analysis is no longer “systematically” done.Ģ. The compensation matrix should not be touched at all. 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.

Flowjo tutorial compensation