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Writer's pictureolivertburton

Blocking: Tandem breakdown

Updated: Oct 12

The issue

Breakdown of protein-based tandem dyes, causing a change in the light emitted and consequently, erroneous results.


The reagent

Tandem Stabilizer. Available from BioLegend.


What is it?

I don't know. This paper suggests that Vitamin C can do the job. I may have to try that.


Alternatives

A dedicated red-light room.


Joking aside, I'm not aware of any commercial alternatives. There are several easy things you can and probably should do to reduce tandem breakdown in your experiments. To understand what you can do, it's important to know what's causing the tandem breakdown in the first place. The most important factors are light, temperature, time, fixatives and the cells themselves.


Light exposure: Work quickly when the tandem fluorophores or tandem-stained samples are in the light. Cover your samples as much as possible with something like foil. Prepare master mixes in dark tubes or wrap the tube in foil.


Temperature: Work cold, if possible, consistently if not. Keep mixes and antibody stocks cold, especially in the light.


Time: Older vials of tandems will be more degraded. Mixes containing tandems will likely degrade faster than the stock vial (unless tandem stabilizer is added).


Fixatives: Know what your fixative does to your tandem(s). Formaldehyde-based fixatives will generally cause noticeable but manageable tandem degradation in proportion to the amount of time and temperature for the fixation period. Other fixatives like methanol may degrade the protein portion of the tandem dye, leaving only the acceptor for a very different spectral signature (credit: Anna Belkina, Babraham Spectral Symposium).


Cells: Tandems can be degraded by cellular enzymes. The more metabolically active cells will degrade tandems more. Cells that internalize tandem conjugates will probably degrade them more. So in general, myeloid cells such as monocytes and macrophages show higher levels of tandem breakdown compared to T cells.


In line with the recommendations on the "Dealing with tandems" post, I would suggest that a large part of dealing with tandems relates to panel design. Try to avoid putting tandem dyes on markers that are co-expressed between cell types that will differentially cause breakdown. I'll show an example of this with CCR2 (expressed on T cells and monocytes) in PE-Cy7, but you can also see this effect in OMIP-102 where HLA-DR has been used on PE/Fire 810. If you do that and you don't adequately control the breakdown, you'll end up with variable breakdown between cell types, making it impossible to accurately unmix that fluorophore for all of the cells in your experiment.


Critical: Match light, temperature, time and fixative exposure of single colour controls with that of the samples or you will not achieve correct unmixing/compensation. If you are staining a cell population that causes tandem breakdown with a tandem, use cells as the controls.

Critical: As much as possible, avoid combining multiple factors that cause tandem breakdown.


What it’s supposed to do

Block tandem breakdown.


Examples

Does it work? Yes, yes it does. Here are some examples from human PBMCs. These have been stained for an hour at room temperature (in the dark), fixed for 30min (at 4C) and incubated overnight at room temperature (in the dark) in perm/wash buffer. I've prepared all the master mixes in dark tubes and kept things in the dark as much as possible, but as you'll see, there's still breakdown.


To visualize this easily, I've not stained for anything in PE or APC. Any signal there is from breakdown.


I've added in the tandem stabilizer as recommended for the last wash of the whole protocol and in the buffer the samples are stored in for acquisition. As that seemed inadequate to me, I've also compared adding the stabilizer at various earlier points in the protocol, including up to all the way through all incubation steps.


Staining note: TIGIT, CCR4, CD95, CCR2 and Integrin beta 7 are being stained on the surface here, prior to fixation.


Unmixing note: Unmixing is being performed using controls prepared with tandem stabilizer added throughout. Thus, the unmixing is intended to be correct for stabilized samples and to allow us to see the breakdown.


Looking first at monocytes, we can see that adding the stabilizer throughout generated the best results. Note that breakdown doesn't just manifest as a compensation-like skewing error. In this case, there's breakdown generating false positives (as well as a minor skew that we'll see below). These sorts of errors are harder to catch in a fully stained sample.


There are similar effects with other tandems. The data are cleaner with the tandem stabilizer added. Note that the CD137 staining here is likely non-specific.



By gating to CD4 T cells, the data are much cleaner even without the stabilizer. However, we can still see improvements by adding the tandem stabilizer. This comes in two forms: First, the upward skew towards the parent molecule (PE, APC) indicative of generalized breakdown, and second, the insidious fuzz of false positives connoting higher amounts of breakdown on some cells. The skew is most evident in PE/Fire 810, PE-C7 and APC/Fire 810, but there's not a lot here because I've tried to limit this with the techniques mentioned above.


Any detrimental effects?

None that I have been able to observe.


How much to use?

The recommended use is a 1:1000 dilution of the stock. This works well. I have not tested anything higher than this. At lower concentrations the effect tapers off, so I would not recommend using less than 1:2000. If you're working with tumours or other tissues with lots of activated myeloid cells, it's possible you'd want more.


When do you really need it?

I would recommend using it any time you are using these fluorophores:


  • PE-Alexa Fluor 610

  • APC-Cy5.5

  • APC-Cy7

  • PE-Alexa Fluor 647

  • PE/Fire 810

  • PE-Cy5.5

  • PerCP-Cy5.5

  • APC/Fire 810

  • PE-Cy7

  • PE-Vio770

  • APC-eFluor 780

  • PE-Vio615

  • -----big gap-----

  • PE-eFluor 610

  • PE/Dazzle 594

  • PE-CF594

  • PE-Cy5

  • PerCP-eFluor 710

  • PerCP/Fire 780

  • PerCP/Fire 806

  • PE/Fire 640

  • APC/Fire 750

  • PE/Fire 700

I've listed from most to least important, roughly, based on how much trouble I've had with them. That experience will be unique to the particular conjugates I've used. If tandem dyes aren't included here (e.g., APC-H7, APC-R700, APC-Alexa 750, PE-Texas Red, PE-Alexa 750), that's because I don't use them.


Bottom line/how to get the best results

Add it to your master mix and to the buffer you store the samples in prior to acquisition. If you're doing intracellular staining, you can add it to your intracellular master mix as well and can try adding it to the fixative.

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