u/Responsible_Rub8057

https://preview.redd.it/i4ptcc1y3h2h1.png?width=1344&format=png&auto=webp&s=b37b26c778fb1e0cfb91326cfcac3e891bddbe78

Hey everyone,

I'm having some trouble approaching my gating to get the "clean events".

Specifically, aside from clear debris (low SSC/FSC, non-specific binding), I have one population that is slightly lower than lymphocytes on FSC, about the same on SSC. I am expecting a lot of immature cells in my samples (lower FSC), but also there is a lot of dead cells (also expected). When plotted against viability, this lower FSC population sometimes shows increase in viabilty dye binding, with a specific population in what i would consider "dead" cells region - but not always! Some plots as an example (singlets removed, gated on CD45+ events). This is consistent across all my samples, regardless of sample quality (as you can see, it's quite variable).

I could use some advice on how to approach this. My current train of thought is to assume that it is immature cells and include them in the lymphocyte gate, but exclude this population in the dead cell region with the assumption that it's early apoptotic cells (keep the boundary, whether it is there as a clear population or not).

Thanks in advance!

Hey folks,

How do you handle flow data (frequencies of parent) when you have to apply some linear models after? I've been told that, because the data is compositional, it cannot be used for, e.i linear regression, and needs normalisation (specifically the person suggested central log normalisation). In my trainings so far I havent heard if this being done, but it is true that maybe I haven't been paying enough attention with "I'll cross that bridge when I get to it" mindset. Well, I've reached that bridge.

I need some thinking and googling time to really understand why it's so, but in the meantime I'm wondering how do you all approach (lets use big words) "data science" on flow end outputs (freqencies). I see it would be different when using MFIs - I dont plan on using this type of output, but if you have some suggestions how you approach MFI output as well, I'd appreciate it.

Thanks!