OpenFlow: Three Colour Immunophenotyping and cell cycle analysis

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In this session we will look at the cell cycle profiles of phenotypically defined subsets using a full spectrum cytometer. This will pull
together some of the things we have looked at in previous sessions - the importance of sample preparation, consideration of
fluorochromes, and controls. We will look at the set-up of the cytometer and the plots and gates needed to produce optimal results. We
will be using human blood cells and a Cytek Aurora.

Опубликовано:

8 сен 2024

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Комментарии : 3

@UChicagoFlow Год назад

59:51 - I had an argument with Cytek people over this. My point is that changing a handful of detectors' gains would change the spectral profile of the fluorophores, but all things being equal, shouldn't change the analysis in the end. Unless it prevents from resolving specific pairs of fluorophores that are so similar that they shouldn't be in the same panel to begin with. Haven't got around to testing it out. The advantages of my way of modifying the gains (over Cytek's preferred way of reducing all gains of an array by the same percentage) are non-existent. But I'll die on this hill. Well, I'll catch a cold on this hill.

@OpenFlowCytometry Год назад

Hello! Kathy here. I do understand what you are saying here in terms of lowering individual detector gains. I think from an end user perspective there is a danger with starting to adjust individual gains. If you are an SME and want to play around with individual gains vs. gain reduction for the whole laser line, go for it and see what yields best results! For a user of the technology that is learning, I think there is a lot that can go wrong with individual gain reduction, especially as you are not accustomed to this workflow. In the case of DAPI, it is usually screaming screaming bright on UV. If you are in a scenario where you can't titrate down the concentration of the dye being used, because it is so bright off of so many detectors, I am in favor of reducing the full set of ADPs. I do understand what you are saying though! It would be fun to test out a whole of different scenarios. :-)

@RuiGardner Год назад

This is a great question, to which I don't have a definitive answer to, as we haven't tested different scenarios in a systematic way. But think of it this way. With spectral flow cytometry the fluors are identified and even distinguished based on the proportions of signal between each detector, i.e., their spectral signatures. Let's say you have two red dyes, F1 and F2. The first one peaks in the first red channel (R1) then drops almost to zero in R2 and the other is the opposite. If you reduce R1 gain significantly, you din't really impact the signature of the second fluor (F2), but you did impact significantly the capacity to measure F1. And maybe someone can correct me if I'm wrong, but in this scenario I would say not only you limited the chances to quantify F1 (you reduced significantly the channel that was providing most of the information to detect F1), but you've also changed the similarity between them (to better or worse). So it also makes it hard to predict whether a panel will work or not if you later change those proportions. I may be wrong, but without knowing enough, I would suggest maintaining the proportions/signatures and just changing all the channels proportionally rather than playing around with the gains individually.