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Issues staining with IgA_148Nd (polyclonal) from Fluidigm

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jcvillasboas

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Post Mon Feb 26, 2018 4:31 pm

Issues staining with IgA_148Nd (polyclonal) from Fluidigm

I am seeing a very weird pattern when staining PBMCs with the IgA_148Nd (polyclonal) from Fluidigm (see picture). What you see here is the result of incubating 1 mCL of stock with 1.5 x 10^6 cells in 100 mCL of total volume. The gate is live intact singlets. No formal titration done yet. Any input from other with experience with this product or other anti-IgA clones would be greatly appreciated. Best. JC
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mleipold

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Post Mon Feb 26, 2018 5:00 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Hi JC,

What are you finding to be weird about that staining pattern? It looks over-titered to me, but otherwise OK.

I have attached a PDF of IgA staining for 3 different donors, Live Intact Singlets and B cells. I am using an in-house conjugate using BD's monoclonal Clone G18-1. If anything, I could afford to decrease my own titer slightly.


Mike
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nlazarus

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Post Mon Feb 26, 2018 7:31 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

JC,

I have had issues with IgA-148 too in that the staining was consistently inconsistent. I tried several other anti-IgA antibodies and found one from BD that worked: G18-1 This is the same clone that Mike is using. I conjugated it successfully to several different metals and it has worked well.

Hope that helps,
Nicole
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jcvillasboas

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Location: Rochester - MN

Post Wed Feb 28, 2018 2:40 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Thanks Mike and Nicole. I think it may be really just a matter of over titration although I wonder if the fact that I stained the sample for both surface and cytoplasmic IgA had something to contribute to the staining intensity being so much off from the other markers. I will stick with this clone for now and try a formal titration. If that does not work I will give a try with G18-1. Thank you much.
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jcvillasboas

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Posts: 41

Joined: Fri Apr 03, 2015 3:22 pm

Location: Rochester - MN

Post Fri Mar 02, 2018 5:08 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Following up on the staining pattern using polyclonal anti-human IgA (on 148Nd) from Fluidigm. This week we ran a formal 4-point titration curve by staining the same 1.5 x 10^6 PBMCs with 1X (1 mcL of stock per 100 mL of staining volume), 0.5X, 0.25X, and 0.125x. As Mike suggested, I was certainly overstaining but I am still getting non-specific staining at low titers (this time I included CD3 as a way to monitor T cells [manually gated as CD20-CD3+ live singlets] for non-specific binding). Snapshot included below:

Polyclonal IgA titration curve.PNG



I have also attached a PDF with an overview of all the channels that contribute signal into 148Nd (stratified by main populations). My interpretation of the data is that the signal on 148Nd seen in T cells can only be explained by non-specific of the antibody (and not from spurious signal). I don't know why but the amount of non-specific binding seems to increase with less antibody. I thought it could be the way I am gating the IgA+ cells but that seems true even if I set the gate using a biaxial plot of IgA vs IgD using B cells as reference:

IgA vs IgD.PNG


Can I pick your CyTOFer brains here? Am I coming to the right conclusion? Am I being too strict? Is this amount of non-specific binding acceptable and comparable with what is seen with the other clones? Should I just keep going down on my antibody concentration or is it time to abandon this clone and try a new one (G18-1 perhaps)?

Thanks again in advance for all the help.

Best

JC
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Signal coming into 148Nd - stratified by populations.pdf
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mleipold

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Post Fri Mar 02, 2018 5:40 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Hi JC,

The background nonspecific binding does seem still a bit high. I know that I had to really titer my IgA and IgG, so it might be worth going down even further.

As comparison, here is IgA vs Ir for the major lineages in my control cells (G18-1 monoclonal from BD; btw, Nicole was the one who recommended that clone to me). I have a couple high dots in my CD8+ T cells, but not enough that I'm worrying about it....definitely fewer than what you're seeing.


Mike
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IgA vs Ir.pdf
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jcvillasboas

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Location: Rochester - MN

Post Fri Mar 02, 2018 10:58 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Thanks a lot Mike. Looking at your data it is clear there is work to be done. A follow-up question regarding these titration procedures: once you define the optimal concentration, do you typically dilute the stock vial into working solutions (by diluting the antibody so that you always have to use, let's say, 1 mcL per 100 mCL of staining volume)? If so, do you just use the typical antibody stabilizer solution with Na azide for that dilution? Thanks.
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mleipold

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Post Fri Mar 02, 2018 11:25 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Hi JC,

Personally, I don't make a further dilution of my stock, aiming for 1uL/test or whatever. I let Excel tell me how many microliters I need for a given number of samples I'm staining on the same day.

The only exception to this is when I have such a small volume of antibody that I don't feel that I can't accurately pipet it....like, 0.1uL total for 10 samples, or something. And yes, in that case, I use the same stabilizer+azide.


Mike
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auyeunga

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Post Wed Apr 11, 2018 11:18 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Hi Mike,

Was it a surface stain you did for the IgA (G18-1) clone?

Thanks!
Amelia
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mleipold

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Post Wed Apr 11, 2018 11:30 pm

Re: Issues staining with IgA_148Nd (polyclonal) from Fluidig

Hi Amelia,

Yes, I did surface staining for the IgA. Cryopreserved PBMCs, thawed, washed twice with cRPMI+benzonase. I did CD45 barcoding, then surface staining, so the cells were live.


Mike

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