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Staining/compensation problems with aqua live dead

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#1 calistan



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Posted 04 September 2013 - 11:12 AM

Hi, I'm new to the forum and hopefully I'm listing everything correctly.

I'm a graduate student working on obesity.

I'm running a Phenotype panel on an LSR II Custom flow cytometer.

I'm using facsdiva software

and analyzing using flowjo.

I've been using aqua live dead to try and try to clean up my images when I run my samples. Which for the most part this has been working well on samples that weren't frozen down ideally. However when Ive been running a non lymph panel to look at monocytes, DCs, and NK cells, the "aqua" has been looking strange.

My panel consists of

  • Aqua live dead
  • CD3/CD20 same color
  • CD16
  • CD11c
  • CD14
  • CD123
  • CD56
  • HLA-DR

When I've been gating my NK cells (off of aqualivedead- and then nonlymphgate, then CD3-CD20-HLADR-CD14-) I've had  little to no CD56bright cells. However when I manually adjust the transformation of the amcyan (aqua live dead) I see a population outside of what was originally gated. When I gate on this alone, I have a large population of CD56bright ONLY. So I'm curious if anyone has had this experience or can offer an explanation? or if my gating choices are wrong. Hopefully the image will help. In the image I've gated with/without the population.Attached File  Document5.pdf   700.72KB   498 downloads

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