And we're back! Flu season is a doozy.
One question we got asked after publishing this was whether we had tried it with tetramers. I thought it would work because tetramers are often low affinity, and low affinity antibodies often show greater improvements with overnight staining. Also, problematic staining combinations such as TCRgd with CD3 on human cells become unproblematic with overnight staining, presumably due to the small fluctuations in the complex over time that allow antibodies access, overcoming steric hindrance. These things suggested that overnight incubations could help. That is not the case, as far as I can tell. I wasn't able to address this before because we weren't doing a ton of work with tetramers at the time.
For some background, I recommend reading these articles from Andrew Sewell's group on getting the most out of tetramers:
You can also see some nice examples of how different times and temperatures may affect staining from the NIH tetramer core.
The important things we already know for working with tetramers include the following:
Spin the vial before use to remove aggregates.
Use dasatinib to prevent TCR internalization as long as you are not performing functional assays.
Use bright fluorophores.
Test different times and concentrations on your cells to find the optima.
Use higher valency reagents, such as dextramers, to enhance detection of low affinity TCRs.
Design your panel and protocol to give clean space in the tetramer detection channel. For example, using the tandem stabilizer will help prevent "FACS dust" from PE-tandems into the PE-labeled tetramer. Avoid co-expression of spread-causing fluorophores on the same cell type that your tetramer will be on.
Using amplification, such as anti-PE, requires designing your panel not to contain any other fluorophores with PE in them. Getting these amplification steps working well takes some effort and probably isn't for most users.
Right, now, let's get back to the question at hand.
Here’s an example of staining a MOG tetramer (on PE) on TCR transgenic mice. This is a lowish affinity TCR. The extended incubation doesn’t help, while raising the temperature does. This is on the surface of the cell, in the presence of dasatinib. The cells have been gated on viable CD4+CD3+ lymphocytes.
The best detection occurs at room temperature. Slightly better results were obtained by extending the incubation for 2hrs.
Let's look at an example from the other end of the spectrum of affinity: the iNKT TCR. For this, we've got the mouse CD1d tetramer loaded with the PBS-57 high affinity ligand. For this tetramer, the best results I get are at 4C. Higher temperatures seem to be either causing TCR internalization or, probably, increased death in the iNKT cells.
Fixing and permeabilizing also does not work for tetramers from what I've seen so far. For the CD1d tetramer, we see a massive increase in background that is present in both the PBS-57-loaded and control tetramers. This is what I've seen so far with three different mouse tetramers. I’m guessing this is because these reagents are quite large and are hard to wash out of the cell. Perhaps it would work with a different type of blocking, using unconjugated control tetramer.
In any case, for now, our best approach for standard flow analysis is as follows:
Titrate on a sample containing a decent population (ideally > 0.5%) of positives.
Stain for 2hrs at room temperature in the presence of dasatinib.
Wash, fix.
If you're doing functional assays such as degranulation, AIM or cytokine staining, you'll want to leave out the dasatinib. While it may be tempting to incorporate the tetramer staining into the 37C incubation stages of functional assays, I'd recommend against it due to 1) the azide in the tetramer, which will reduce functional readouts, 2) degradation of fluorophores such as PE and APC at 37C and 3) potential for tetramer-induced TCR signaling.
Say's Phoebe, Oregon, USA
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