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EQ beads positive

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Marjolyn

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Posts: 9

Joined: Tue Sep 15, 2015 6:02 pm

Location: Leiden, The Netherlands

Post Thu Jun 22, 2017 7:09 pm

EQ beads positive

Dear all,

I added a small presentation about a problem I'm facing.

I stained negatively isolated (human) Bcells, first with tetramers labeled to PE and APC. After that I came back with anti-PE and anti-APC and a few other lineage markers. When analysing the data I found out that there is quite a high PE signal. This was caused by the EQ beads that we highly positive for PE and not for APC (same tetramers).
I understand that the beads-cell doublets are not unusual, but what worries me is that it is specific for certain markers. The beads where also positive for a few lineage markers but not all.

Did anyone else noticed that some markers bind more frequently to EQ beads then others? And is there an explanation for that? Is PE more sticky than APC?

Thanks in advance,
Marjolijn
Attachments
Troubleshooting Beads.pptx
(1.81 MiB) Downloaded 367 times
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avinash1

Participant

Posts: 16

Joined: Fri Jun 16, 2017 1:59 pm

Post Fri Jun 23, 2017 1:47 pm

Re: EQ beads positive

Looks like you are having M+16 oxidation spill over between 140 Ce beads and 156 channel. Remove the beads and then look at the data !!
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Jun 23, 2017 6:27 pm

Re: EQ beads positive

Hi Marjolijn,

I suspect that your tetramers are binding to the beads non-specifically. These type of beads will readily bind any protein, so if there is any free protein in your sample it will stick. This may be because the tetramers have a higher off rate and so come off of your sample to a greater extent than the other antibodies that you're analyzing. Obviously, the anti-APC and anti-PE antibodies themselves are also proteins and could be binding directly to the beads or binding to the beads while bound to your tetramers. I don't have a great explanation as to why PE binds better than APC, but I doubt it matters that much.

I'm also not as sure why your lineage markers are getting stuck to the beads, I don't think I ever seen this. I would try to be sure that you're washing your cells thoroughly after antibody binding and that your post fixation (typically during intercalation) is adequate. If you're only seeing the lineage markers on the beads (i.e. no Ir signal) then I would think this could only be coming from free antibody that has fallen off of your cells. You could try saving the supernate from each of your post-staining washes, then diluting and running it in solution mode (you'll have to empirically sort out the right dilution, but it will probably be a low nanomolar concentration of staining antibody). This should allow you to directly see how much antibody is falling off of your cells at each step of your protocol.

Fortunately, the solution to your problem should be easy. I would suggest that you try pre-washing your EQ beads in a protein containing solution (e.g. cell staining media) then wash a few times in water, then add them to your cells. I would bet that this will greatly reduce the non-specific binding that you're seeing. But it would be nice to do the experiments above, as you'd probably like to sort out why so much of your antibody is falling off your cells.

Let us know what you find out.

best,

Greg
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Marjolyn

Participant

Posts: 9

Joined: Tue Sep 15, 2015 6:02 pm

Location: Leiden, The Netherlands

Post Mon Jun 26, 2017 8:59 am

Re: EQ beads positive

Dear Greg,

Thanks for your response.

First to reply to avinash1, ofcourse I gated out my beads, but it was surprizing how much difference there was in signal of the anti-PE and anti-APC. That was the reason why I was searching in my gating strategy to find out where I was loosing signal. Gregs option could be the solution. I will have a try with pre-washing my beads, to see if the PE signal could be increased a bit.

Keep you posted!

Best,
Marjolijn
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jun 26, 2017 2:48 pm

Re: EQ beads positive

Hi Marjolijn,

When you were running your samples, was there much streaking in the lineage and PE-tetramer channels?

The reason I ask: since the software grabs the time-window defined by a cell event and calculates the signal intensity in each channel, signal coming from a streak winds up being included in the FCS file for that cell event. Unless you're taking notes during acquisition, it can be difficult during later data analysis to figure out what is originating from a streak (debris, overtitered antibody,e tc) and what is coming from punctate cell-associated signal (either specific at high or low signal, or nonspecific binding at low signal).


Mike
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Marjolyn

Participant

Posts: 9

Joined: Tue Sep 15, 2015 6:02 pm

Location: Leiden, The Netherlands

Post Wed Jun 28, 2017 8:40 am

Re: EQ beads positive

Hi Mike,

Thanks for your input.

What I remember is that the rainplot looked normal. No weird streaks .
I think the big problem is the crosstalk from the 140Ce channel. Every bead will be dim positive for 156Gd. This will be the case for all the beads, that’s why the whole population is dim positive for the 156Gd channel.

The PE signal does not get higher with Beads+DNA+. Therefore I think the low PE signal probably comes due to competition with the other tetramer. Maybe the APC had a little bit higher affinity, both tetramers have a different dilution (optimized with FACS).
I will optimize the experiments further.

Do you ever used secondary antibodies in CyTOF? Fluidigm has a few secondary antibodies available like anti-PE, APC/FITC/GFP and Biotin. I would like to conjugate a secondary antibody myself. I think this works the same as conjugating a primary antibody. I was wondering if anybody has any experience with that?

Best,
Marjolijn
Attachments
Beads.jpg
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DMcDonald

Contributor

Posts: 21

Joined: Thu Nov 26, 2015 10:14 am

Post Wed Jun 28, 2017 1:26 pm

Re: EQ beads positive

Hi,

Have you ruled out oxidation? 156 is +16 from your EQ 140Ce. How about the other slightly positive channels you see unexpected signals in; are these +16 away from the EQ isotopes?

Best wishes,
David
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RuthCyTOF

Participant

Posts: 7

Joined: Sat Nov 30, 2013 10:19 pm

Post Wed Jun 28, 2017 2:18 pm

Re: EQ beads positive

Hi Marjolin
We have labeled cells with an unconjugated primary antibody and used a Fluidigm-conjugated anti-FITC (or other fluor) and seen good signal. It's an easy way for new users to try CyTOF without purchasing a lot of reagents.
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dtdavani

Participant

Posts: 12

Joined: Fri Sep 11, 2015 9:52 pm

Post Wed Jun 28, 2017 4:04 pm

Re: EQ beads positive

Hi Marjolin,
We conjugate and use anti FITC, anti-GFP ( IgG1), anti PE in a regular basis and have a good signal. Here is an example of metal tagged 145Nd anti-PE titratio.
antiPE.tiff

I hope this helps,
Dariush

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