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Non-specific extracellular staining despite FcR blocking

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cchedid

Participant

Posts: 4

Joined: Tue Jan 22, 2019 3:17 pm

Post Fri Feb 01, 2019 10:36 am

Re: Non-specific extracellular staining despite FcR blocking

Hi all,

Thanks Mike for pointing out the upload attachment button I somehow managed to miss... classic. So attached is a PDF of cleaning strategy I've been using (following Fluidigm guidelines on Gaussian parameters gating, also included Ir vs EL) and examples of possible nonspecifically stained populations.

@greg, thank you so much for your response. Definitely very interested in the heparin block and reading Adeeb's paper ASAP! Also the attached file might help explain what I mean when I said all of the strange double positive populations disappeared after one of them was gated out.
Regarding my protocol, my cells are fixed immediately after collection from the blood, then frozen. Later on when I perform the staining, I thaw the cells, stain the extracellular markers, then barcode following classic Fluidigm protocols, and finally stain the intracellular markers.

@Christoph (with no e), I considered just gating out the implausible cells and analyzing the rest from there but I'm worried that if it indeed is nonspecific staining, too many antibodies might be "sucked into" those implausible populations and cause loss of resolution for less frequent populations (i.e. gamma delta, MAIT cells). Does that make any sense?

@Mike, I was running them at around 0.8M/mL but definitely might be worth trying out dilutions and see if it helps. Thanks a lot for the paper!

Cheers, Carole
Attachments
01-Feb-2019-nonspe staining.pdf
(1.23 MiB) Downloaded 119 times
PhD student at International Center for Infectiology Research (Lyon, France)
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 01, 2019 6:38 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Carole,

The first thing that jumps out at me is that you're trying to gate Event Length while using a Log (or Arcsinh) scale.

You need to switch your EL parameter to Linear; this will give you much greater control over your EL gating. I typically have EL scale ranging 0-80 during gating.


I think you said that you've been using a Helios for acquisition. If so, the Ir-bright singlet "hotspot" for EL is usually ~17, so doublets are going to start near EL=34-35. I usually gate pretty tightly on this for singlets, so I only take about EL=10-30 (EL=10 being the default lower limit for Helios acquisition). That would be a really difficult cutoff for Log scale.


Mike
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