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CD signal decreasing over time...

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Jahangir

Contributor

Posts: 34

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Jul 23, 2019 2:08 pm

CD signal decreasing over time...

Hi guys,

Hope everyone is well.

So, my colleague is running some human gdT-Cells. The attached screenshots are all ungated biaxials for:
- CD45 (HI30) against time
- CD25 (2A3) against time
- CD44 (IM7) against time

But as you can see, the signal slightly decreases over time... has anyone got any suggestions why this is occurring?

It's only happening with these specific antibodies and not any intracellular antibodies.

Many thanks,

Jahangir
Attachments
Screenshot 2019-07-23 at 14.59.59.png
Screenshot 2019-07-23 at 15.05.25.png
Screenshot 2019-07-23 at 15.05.10.png
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eganio

Participant

Posts: 10

Joined: Wed Nov 07, 2018 8:00 pm

Post Tue Jul 23, 2019 3:53 pm

Re: CD signal decreasing over time...

Hi Jahangir,

Did you use normalization beads and normalize the data post-acquisition? Your plots look very similar to non-normalized data, although you would think all of your Abs would look like this if that was the case.

-Ed
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EHaasDFCI

Participant

Posts: 19

Joined: Thu Jun 15, 2017 5:34 pm

Post Wed Jul 24, 2019 6:40 pm

Re: CD signal decreasing over time...

Hi Jahangir,

Do you happen to have any markers in the 89Y channel? Were these run in CAS or water?

Eric
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Jahangir

Contributor

Posts: 34

Joined: Sun Oct 29, 2017 6:34 pm

Post Wed Jul 24, 2019 7:09 pm

Re: CD signal decreasing over time...

Hi Ed and Eric,

Many thanks for your replies.

Ed:
- I did use normalisation beads and I did normalise the data post-acqusition. I normally add EQ beads to a ratio of 1:5 (EQ beads:water).
- And yeah, a lot of my intracellular markers do not have the same pattern at all.

Eric:
- I did have a marker in the 89-Y channel. It was the CD3 (UCHT1 clone), which I conjugated myself. I know the polymer chemistry isn't designed to be used with the Yttrium metal, but it actually works brilliantly.
- It was run in water.

Any thoughts?

Best regards,

Jahangir
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technolust

Participant

Posts: 4

Joined: Mon Jul 13, 2015 9:07 pm

Post Thu Jul 25, 2019 1:21 am

Re: CD signal decreasing over time...

I believe that level of change in signal over time reported in this thread by Jahangir is larger than the signal change one typically sees (prior to normalization).

I'm attaching examples of CD45 on 154 and beads on 140 over time from pre-normalized files from a series of 6 samples (from a larger set) ordered by time of collection in the day (left to right / first to last). The bottom and top plots are the same data; the bottom has flat lines through the plots as a reference point (pink). The signal is fairly stable within a sample over the short term (0.7M time) and even over the start to finish of this longer run, and these small differences are addressed by normalization software. Note each sample is a different individual, so we can't compare the CD45 from the starting sample to the end sample, but within a given individual it was relatively stable.

Bead normalization addresses the small difference in signal between the starting and ending samples. In other words, these are pre-normalization and pre-"single event gating" so that Jahangir can compare. Analysis should normally take place after bead normalization and after single event gating. After normalization these data looked great.

2019 signal over time pre-norm on six different samples.jpg


I hope this helps!,

-Jonathan Irish @ Vanderbilt
Last edited by technolust on Thu Jul 25, 2019 4:18 pm, edited 1 time in total.
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Jahangir

Contributor

Posts: 34

Joined: Sun Oct 29, 2017 6:34 pm

Post Thu Jul 25, 2019 11:02 am

Re: CD signal decreasing over time...

Hi Jonathan,

Many thanks for all the information you provided. However, from your attachment, the CD45 antibody is in the 156-Gd channel rather than the 89-Y channel. But nevertheless, it shows that the signal doesn't really drift that much compared to what I observed. I'm still trying to figure out why I observed those results. I'll update everyone if I (ever) find out.

Best regards

Jahangir
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technolust

Participant

Posts: 4

Joined: Mon Jul 13, 2015 9:07 pm

Post Thu Jul 25, 2019 4:42 pm

Re: CD signal decreasing over time...

Thanks Jahangir for catching my typo! I edited my post. (I had another example I was going to post originally.)
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datamanifold

Participant

Posts: 1

Joined: Tue Aug 28, 2018 12:49 pm

Post Fri Jul 26, 2019 9:25 am

Re: CD signal decreasing over time...

Hi thought to let you know we had a similar issue with one of our antibodies when we were running some samples.
At that time we concluded we left the sample in water longer than usual (to deal with some blockage). Not sure if that is the case with your sample..? Unfortunately if this happens the signal is no longer reliable and we ended up not using that sample.

Thanks
Eleni

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