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p-STAT5 background

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ianfrank

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Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Thu Feb 06, 2014 11:20 pm

p-STAT5 background

Dear All,

I'm almost ready to go with a panel, but there are a few background issues that I'm concerned about. I've attached a pdf of the dot plots from FlowJo. In the p-STAT5-150Nd channel (purchased from DVS) There is quite a bit of background further up the axis from the real events. I don't think these are real as they don't appear in the FC data collected the same day. The gated MC events correlate quite nicely with the FC events in the frequency and intensity. Possibly the new lot of pSTAT5? Is there some other source for the background? :?: Also CCR7-166Er is showing some background. This is one that I've conjugated with the Maxpar kit to R&Ds clone 150503.

Ian
Attachments
background.pdf
(122.47 KiB) Downloaded 435 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 07, 2014 12:48 am

Re: p-STAT5 background

Hi Ian,

I'm having trouble following your gating strategy.

I wouldn't gate as broadly as you do on the Cell length: I usually keep in the 20-40 range, maybe go up to 50. But never above 60 (2x30, where 30 is where the main hotspot usually is for my cells). So, I think you have a number of doublets included.


Even putting that aside, I can't follow your plots. With Ce as your y-axis in the top middle plot, your circle is around the calibration beads....are you doing some sort of "everything except what is circled" gate?

Going from top middle to center left ("beads" population....), your event count increases? From the file names, it looks like top middle to center left, you switch samples. Based on your CD4 plots, I think the 20140129_144607 file is your stim'd sample. Do you see this CD3- CCR7bri population in the unstim too, or just the stim?

I would agree that it's unlikely that the CCR7bri are oxidation spillovers, since they're not Nd150-pSTAT5 positive as well.


-Mike
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Fri Feb 07, 2014 6:31 pm

Re: p-STAT5 background

Hi Mike,

I've tightened up the cell length gate and attached a new pdf.

You are right about the circle gate. It's a negative gate to get rid of the DVS EQ Four Element beads that should have been gated out by the parent gate. ...beads are sticking to the cells?? The cells were resuspended in a solution of dH20 and beads just before collection. I even used half the recommended concentration.

For CCR7 I'm just showing representative plots of the gating scheme. The actual plots look identical. This sample is stimmed, but the unstimmed still has the same background. I should mention this is stained after the fix. In previous experiments I've stained with CCR7 during the 15 minute IL2 stim. This produced less background, but didn't get rid of it totally.

Any more ideas? I really appreciate the input.

Ian
Attachments
background2.pdf
(125.32 KiB) Downloaded 341 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 07, 2014 6:58 pm

Re: p-STAT5 background

Hi Ian,

I haven't used the beads much yet myself. However, what I'm hearing from regular users of them, the beads stick to cells. You'll want to use the 0.2x bead stock (made that day) to dilute your samples only IMMEDIATELY before filtering and putting on the machine. Do not dilute all your samples with the beads, and then start running them.

But yes, you will need to do a bead exclusion gate. You can do an Ir by Ce bivariate: Ir+Ce- are your cells, Ir-Ce+ are your beads, and Ir+Ce+ are bead-cell doublets to be thrown out of your analysis.


I personally have only been staining live cells. However, Rosemary at the HIMC does phospho (stim, fix, then stain). She has not had luck with CCR7. In her hands, it was too dim to be useful, and had background issues besides. I think she's using CD27 for her Naive/Eff/CM/EM gating. If you have CD27 in your panel, try gating CD27 vs CCR7: at least for T cells, there should be 80+% correlation between those two markers (donor-dependent, and not perfect; some of the intermediate states truly do have just one or the other).

If there's not high correlation between them, then it's a post-fix staining issue. If there *is*, then there is some other problem. But if you have CD27 in your panel, then you have the data already to check.


Mike
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Fri Feb 07, 2014 9:10 pm

Re: p-STAT5 background

Thanks, Mike. Do you know if Rosemary or others have gotten any bad lots of p-STAT5-150Nd from DVS? Could this explain the background?
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 07, 2014 9:17 pm

Re: p-STAT5 background

I haven't heard of any issues. But contact DVS about it....Pam and her reagents team are good about checking on that stuff.
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Thu Feb 20, 2014 10:32 pm

Re: p-STAT5 background

Mike,

I've repeated the p-STAT5 staining mentioned below. I've included the same donor as well as 2 more donors. I did a number of things within this experiment to try to isolate where the background was coming from, but there wasn't any! The only thing that I did differently was to spin down the p-STAT5 conjugate as well as the cocktail of antibodies. Hopefully this will clear it up from now on. Thanks for the suggestion.

The CCR7 problem was not addressed in this experiment. From previous work it was known that the CCR7 background was unrelated to the pSTAT5 background. I'm still looking for a good phosphlow CCR7 conjugate.

Ian
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 20, 2014 11:58 pm

Re: p-STAT5 background

Hi Ian,

Thanks for the update.

We have seen issues with antibody aggregates from time to time. There may be a few antibodies that aggregate more often than others, but we've had a wide variety have aggregates from time to time, prep to different prep.

This is one reason why we put all our antibodies through one of the 0.1um spin filters as the cocktail, immediately before applying it to the cells.

Even then, occasionally there's a particular antibody prep that has aggregates that slip through. You can see them as bright cell-like events during acquisition, where there is no Ir staining, and basically nothing other than that one channel.

In those cases, you did the right thing: centrifuge down the antibody stock and transferring to another tube, or at least avoid pipetting down to the bottom of the tube.


Mike
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Wed Mar 12, 2014 5:43 pm

Re: p-STAT5 background

Hi Mike,

I was trying to find the 0.1um spin filters that you mentioned. I didn't see any that seemed right for this. Can you give me any details on these? Brand? Catalog number? where ordered?

ian
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Mar 12, 2014 5:55 pm

Re: p-STAT5 background

Hi Ian,

We use Ultrafree Durapore PVDF 0.1um spin filters from Millipore. I think we buy them from Fisher.

Catalog# UFC30VV00


Mike

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