OpenFlow: Fluorescence Compensation in Diva

When carrying out multicolor Flow Cytometry experiments, it is common to experience fluorescence spillover due to overlapping emission. In this Open Flow session, we will review in depth the principles behind compensation and how it is carried out on a BD Fortessa analyzer. This will be done utilizing the automatic compensation built into the software and a four color experiment.

[@akritino7437]

Just saying! This is just my point of view! I really love the fact that you all have gathered to explicate everything about Flow cytometry and the flow jo analysis. I really needed someone to explain to me so comprehensively like that before ! Thank you so so much for such an initiative !! I am truly grateful! :D

[@debiprasadpanda1395]

Wonderful presentation. Thanks for detailed explanation. Even after being in the flow field for 3yrs, I learned many new nitigrities.

[@sahartahvili5081]

Thank you so much for all four videos you posted. All to the point and nicely narrated. Hope to see more in your channel in the near future.

[@RuiGardner]

Thanks Sahar. It's so nice to hear that. We're going to continue to provide more educational content. Stay tuned!

[@nanaanya2659]

@@RuiGardner a

[@shokoufehkarimi6460]

Thank you for all the posts.

[@OpenFlowCytometry]

You are very welcome! Thanks for checking it out!

[@matthewbell7386]

The concept that compensation controls should be treated the same way as experimental samples (e.g. having compensation controls and experimental samples on the same plate during the staining process/at least running them through the same staining protocol) makes sense to me. A quick question on this, though. If you are fixing your experimental samples and then running them a couple of days later and are using beads for compensation controls, would you still recommend running the beads through the staining process on the same day as the experimental samples and then letting the beads sit until you can run the experiment, or would this lead to a significant loss in the brightness of the compensation beads and lead to compensation issues? Thanks!

[@RuiGardner]

Hi Matthew, that's a very common question. I guess the way to think about it is, if there's anything that can affect your data, you need to control for it. You are correct, fixation can reduce the brightness of fluors. This won't be a problem per se, unless the loss of brightness is such that you no longer have a good separation. But in this case, you would also see that kind of loss in your sample. Since we need to make sure that the signal in the beads is brighter than in the sample, I would be more worried about loss of resolution in the sample. The biggest problem is that fixatives can degrade (or at least change) the spectral properties of fluors, for instance, through changes in pH, crosslinking of peptides, etc. So it would be ideal to have the beads go through the same fixation protocol, including how long they will be exposed. Having said that, if from your experience, you don't see any changes in the amount of compensation/unmixing in your samples when using "fixed" beads made on the same day vs made on the day you fixed the samples, then you can start making them on that same day. It may depend on the fluor, so I would test all of them a few times to give you confidence. But remember, every time you drop a control, you're no longer in control! ;)

[@pingxue6245]

I noticed that your single color negative very close to the left,

[@kathydaniels1077]

Hi Ping! The negative does not matter here. It matters that the positive fully stained experimental sample is on scale and then we want to run the compensation controls at the same voltage. We go into a lot more detail on optimizing voltages in another session!