Tips & Tricks: Temperature and time

Increasing staining time helps with detecting markers in many cases. What about temperature? Some protocols, notably human chemokine receptors and tetramer staining protocols, use 37C incubations to achieve better staining. Let's look at how that works and what the tradeoffs are.

Why would temperature matter? Temperature is essentially the average speed of molecules, so higher temperatures mean the molecules are moving around faster. The more movement, the more likely you are to have collisions between the antibody and antigen, leading to productive binding. There are additional effects on membrane fluidity and protein conformational flipping that can lead to epitopes being revealed at higher temperatures. Steric hindrance can also be reduced by temperature--if you have a big complex of proteins, at higher temperatures the individual elements might shift around more, allowing antibodies access to different portions over time. In this hypothetical example of a protein complex, at lower temperatures, a higher fraction of the complexes would likely never adjust to that antibody-accessible arrangement within the staining incubation time.

Okay, let's look at an extreme example of this: human CCR7 staining. CCR7 is exceptionally temperature-dependent, so much so that if you aren't consistent with temperature control for your staining mix prior to adding it to your cells, you'll get substantial batch effects.

Human PBMCs stained for CCR7 and gated on viable CD3+CD4+ T cells.

Why wouldn't we always stain at 37C, then? After all, this is the physiological temperature (or close to it) for both our cells and antibodies, so it should be where things work best. There are a few reasons.

First, if your cells are not in amazing condition and have been cold, warming them up to 37C will result in cells dying. This is particularly true for cryopreserved cells, like these PBMCs. Note how the monocytes (higher FSC) are more affected.

Human PBMCs incubated under the indicated conditions in FACS buffer, then stained for 10min with ViaKrome 808 fixable viability dye.

That's probably not a deal-breaker for most people.

There are a couple of other problems, though. While mammalian cells and antibodies may be adapted to 37C, the fluorophores are not. Some of these, particularly protein-based fluorophores like PE, APC and PerCP, degrade at higher temperatures. So, you actually get dimmer staining. Tandems will break down faster. In the example below, I'm showing what happens in a longer staining so we can see the effect better. This effect is proportional to the length of incubation, so it doesn't have to be a huge problem with a short staining time. Note that the example below is on fixed cells, so there shouldn't be an internalization involved; internalization of fluorophore conjugates can cause more rapid degradation or pH-dependent spectral shifts.

Mouse splenocytes stained at the indicated temperatures overnight after fixation and permeabilization with the eBioscience Foxp3/Transcription Factor kit. RT = room temperature.

Another issue is that at higher temperatures, the background and autofluorescence tend to increase. If you're staining unfixed cells, the autofluorescence can increase or shift because autofluorescence largely reflects cellular metabolism. If you're staining fixed cells and using a saponin-based permeabilization buffer, like the example below, the prolonged exposure to the buffer at higher temperatures will result in an increase in background, interpretable as autofluorescence. Anything you add to your staining that is visibly coloured (e.g., Brilliant Stain buffer) will do this.

Mouse splenocytes, as above. Unstained.

Finally, cells are active at 37C. They'll change. If you use antibodies that bind to signalling receptors, you engage signalling pathways in the cells. For instance, staining for CD3 or IgM can activate T or B cells, and if left long enough, would cause a change in the distribution of naive vs. activated cells. New transcription will be initiated, leading eventually to new proteins being produced. This probably isn't an issue for most people doing cytometry analysis because you'll be staining for periods of time that are shorter than those required to translate new proteins. It will be an issue if you plan to sort cells and analyze mRNA.

All-in-all, use temperature carefully in your experiments, with an eye to the potential pitfalls. There are some big benefits to be had. I'll leave you today with this final example of CTLA-4 staining after harsh fixation. Staining can be recovered to near-normal levels at room temperature. Personally, I find that room temperature staining offers a good compromise with many of the benefits of 37C staining and fewer drawbacks.