Thank you for checking it out! We are glad to help.

Thanks for the kind words, so glad the video was helpful. I'm not sure exactly where you got confused, but one of the points was that if you assume the drop-charge delay (timing between laser and breakoff point) if you have two cells attached and you don't exclude them, the following scenarios can happen: 1) A negative and a positive cell stuck together will have the total signal of a positive cell (with a little bit more signal from the autofluorescence of the neg cell). So when you sort for positive cells and you don't exclude doublets, those two cells will be sorted together and may split when collected in the tube and you will detect that negative cell when you read the sorted sample back in the instrument. 2) if two negative cells or two positive cells are attached together, then you'll sort those in the respective gates and they won't count for impurities. So if you see a positive cell in your collection tube when you were sorting the negative population, then it can't be because of lack of doublet exclusion because the doublet with a positive cell would fall in the positive gate in the first place. Did this answer your question?

Thanks for your question and feedback. It really makes it worthwhile to develop these initiatives when we see it makes a difference to those watching these videos. As for your question, yes, this is a well known problem with Diva that was never corrected. BD expects their customers to find workarounds for bugs and badly implemented features in Diva. Unfortunately, most of us learnt the hard way, after losing important samples due to this issue.

@@RuiGardner thanks for your answer. Your work is much appreciated! So, do you suggest that we first set all gates with biexponential scale to better see our populations, and then change to log scale when we start to sort? But in this case the gates will still go below zero also on the log scale (if we are sorting a negative population of course). I have never noticed that I have lost cells due to the use of biexponential scale. How can I prove this on my instrument? I think biexponential scale it is almost always used in our instrument.

@@CitometriaCPTUnvr This only happens if you're using biexponential AND all your gate is below zero for one of the parameters. Switching to log scale may be difficult because you may have all your data squashed in the lowest bin. What we typically do is to try and have at least some of the gate above zero, and if that's not possible (maybe the gate is already "touching" the positive populations) we increase the gains to have part of the negative population above zero. This way the gate will also be above zero. You can test this by sorting in biexponential with very low gains so that your negative population is below zero. Then make sure the gate is also below zero and see what happens. It may vary with different Diva versions (although the latest still show the same issue)

@@RuiGardner thanks I will try it. But in your experience, what happens? Does the DIVA software not recognize the cells below zero and therefore the events are aborted? Thanks again for the explanations!

Hi Marta! This is a great idea. Maybe we can plan another sorting class with a few specific applications like single-cell sorting. We'll brainstorm this. :)