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Signal variation upon freezing samples

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Bethmac

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Posts: 2

Joined: Fri Jan 12, 2018 9:26 pm

Post Fri Jan 12, 2018 10:51 pm

Signal variation upon freezing samples

Hi CyTOFers,

We recently tested splitting a stained sample of mouse spleen cells in half and then freezing half (10% DMS0 : 90 % FBS) at -70 degrees while storing the other half (1.65% PFA in PBS) at 4 degrees for just over a week.

We than acquired both samples on a Helios2 during the same run. The proportion of immune cell populations detected was in general comparable between conditions by standard gating. However we noticed that the -70 cells had varying signal over time in almost every metal channel, and, very high background in some channels (e.g. 165Ho TCRb). The worst variation/background appeared in the 165- 169 range.

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Do you think any particular part of the freezing/thawing process could be causing this and is there a way to reduce the variation? Any insight is greatly appreciated!!

Beth
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Bethmac

Participant

Posts: 2

Joined: Fri Jan 12, 2018 9:26 pm

Post Fri Jan 12, 2018 10:58 pm

Re: Signal variation upon freezing samples

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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jan 12, 2018 11:23 pm

Re: Signal variation upon freezing samples

Hi Beth,

When I was doing some initial testing for frozen pre-stained cells, I did find that at least *recovery* seemed to be better if I took the FBS/DMSO tubes, placed them on ice, and let them thaw (takes only about 15min for 100uL FBS/DMSO, I think), then go directly into the MilliQ washes.

This is compared to:
1) using room temp buffer pipetted up and down to thaw the pellet, or
2) placing the tubes in a rack at room temp and thawing that way.

I didn't notice much *signal* difference, but I didn't exhaustively look for it. But the backgrounds also seemed to be a bit worse *not* thawing on ice.


Maybe give that a try?


Mike
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SamuelKD

Participant

Posts: 5

Joined: Tue Dec 15, 2015 4:42 pm

Post Sat Jan 13, 2018 12:39 am

Re: Signal variation upon freezing samples

Some of the channels look very strange about halfway through your -70 sample. Was there a massive clog of some sort? This is especially noticeable with the B220 channel. Were these files normalized?
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Sat Jan 13, 2018 1:13 am

Re: Signal variation upon freezing samples

Hi all

Beth's samples were run in our Facility. We identified this sudden signal drop in certain channels a few months ago. It is sporadic which makes it very difficult to track but we are confident that it has nothing to do with sample preparation, since we can see it when a single sample is split into multiple tubes and run consecutively - ie technical replicates. It is very odd in that the problem occurs only in certain channels (always the same ones) that are spread across the mass range. Thus clearly not a clog as I think all channels should be equally affected? Even more strangely, some of the affected channels also have EQ beads but the bead signal does not change.

Fluidgim has done several evaluations of our 2 Helios instruments (happens on both of them) which did not yet identify the problem. We are waiting for them to schedule another test which they seem to think will solve the problem. I live in hope.

In the meantime we welcome any ideas the community may have and wonder if anyone else has seen this problem?

Cindy
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Sat Jan 13, 2018 1:52 am

Re: Signal variation upon freezing samples

HI Cindy,

You said " It is very odd in that the problem occurs only in certain channels (always the same ones) that are spread across the mass range. Thus clearly not a clog as I think all channels should be equally affected? Even more strangely, some of the affected channels also have EQ beads but the bead signal does not change."

It can be difficult to use the EQ beads themselves to evaluate: they're already so bright that even a 50% drop in signal would barely shift the peak (though of course calculating MFIs would tell you). Sometimes, you can look at the Ce142 and Lu176 minor isotopes: they're in a range of brightness that at least some of your antibodies would be in.

I would agree, I would expect a clog to affect all channels at least somewhat.


When you said "only in certain channels (always the same ones)", do you see this with different antibodies in those same channels? Like, 165 and 166 were affected: does that happen with those metals regardless of whether they're B220 and TCRb or whether they're CD8, CD20, CD19, CD127 or whatever (in-house conjugate or Fluidigm conjugate, either one)?

That might start helping detangle whether it's some crazy thing happening to the cells with certain antibodies (highly unlikely, I know), or whether it truly is a metal-channel-driven problem.


Mike
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Sat Jan 13, 2018 1:56 am

Re: Signal variation upon freezing samples

Hi Mike

The signal drop happens with certain channels regardless of what marker is in them, happens with both human and mouse cells, and with both in-house and Fluidigm conjugates. So I don't see how this could be an Ab problem?

Cindy
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Sat Jan 13, 2018 2:20 am

Re: Signal variation upon freezing samples

Hi CIndy,

It definitely sounds like a metal/machine problem, *not* an antibody/polymer/etc issue.


Mike
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SamuelKD

Participant

Posts: 5

Joined: Tue Dec 15, 2015 4:42 pm

Post Sat Jan 13, 2018 2:21 am

Re: Signal variation upon freezing samples

It's strange. The way you describe it sounds like it could be something wrong with a detector, but you said it's affecting two separate machines. I'm really curious what turns out to be the issue(s). Could it be something that they both share while running? Electrical? Ventilation? Does this phenomenon occur when both machines are concurrently running, or independent?

Clearly this has gone past the sample conditions question. sorry!
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Sat Jan 13, 2018 4:22 am

Re: Signal variation upon freezing samples

Hi Beth and Cindy,

I would certainly agree with everyone else that this is quite weird. But I would suggest a few things that you may or may not have tried/thought of so far.

1. In my experience freezing always disrupts the cell membranes to some extent, and this would be expected to be most significant when a relatively gentle fixation is used (e.g. 1.6% PFA). I suspect the main difference between the frozen and never frozen samples is that the frozen samples are physically breaking apart is some relatively subtle way (loss of loosely attached surface proteins, increased intracellular/intranuclear permeability, decreased interaction between protein binding partners, etc.). If that were the case, this effect could vary based on protein, epitope and antibody clone so it may not seem to be very consistent. This could also lead to a signal and/or background decrease (due to antigen loss) or increase (due to increased antigen accessibility).
-Regardless of the cause, this should be fairly easy to sort out, as this effect would be dependent on the time the cells have been diluted in pure water. I would suggest repeating the experiment, but this time dilute both the frozen and never frozen cells in aliquots of pure water and let them sit at room temp for different periods of time (1m, 5m, 10m,....60m, etc.). If some sort of physical degradation is at play, the longer the cells sit diluted in pure water the greater the difference will be between frozen and never frozen samples (irrespective of whether or not they are running in the machine).

2. I'm really puzzled when you say "The signal drop happens with certain channels regardless of what marker is in them, happens with both human and mouse cells, and with both in-house and Fluidigm conjugates." Could you provide more information about this?
-Specifically, is EVERY effected antibody different when this happens, or just some are different? (I ask this, because you seem to be having a fair bit of M+1 spillover, so I think most of the TCF1_Er167 signal could actually just be spillover from the "bright" B220_Er166 signal. If that's the case, any "dim" antibody on Er167 would show the same pattern, but only if there is a "bright" antibody on Er166. You might have already checked for this. It also seems to be happening in the most sensitive part of the mass range, where spillovers might be most easily detected.
-Does this happen with pure metal in solution mode, or only with stained cells? If it doesn't happen in solution mode, I can't imagine any way this could be a detector, ion optics, or ventilation issue. If it happens in solution mode, it couldn't be a cell or antibody issue. This test should narrow things down quite a bit.
-What do you run just before your samples? Water, wash solution, or something else? It's hard to reduce the pH of a strong acid by diluting with pure water, so if you run sample too close to wash solution (even with water in between) it will strip the metal out of the chelator and give high background and low signal until the cells themselves buffer the acid away, which could take a while. It could also alter the antibody antigen interaction (again in a antigen and clone specific way). This could also be somewhat metal dependent, as different metals have different affinities for the chelator.
-Because of how the beads are made, they wouldn't show most of these effects (except for the portion of the M+1 spillover that's due strictly to mass tailing), which is potentially consistent with what you're observing.


Please do give us some follow-up. I'm incredibly curious to know what's happening.

best of luck,

Greg

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