OpenFlow: Fluorescence Compensation in Diva 

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

@@RuiGardner a

You are very welcome! Thanks for checking it out!

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! ;)

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!