Today I'm starting a series of posts about how to use overnight staining to improve your flow cytometry. I developed this approach over the last few years in Adrian Liston's lab, and we published the basics of the method last year in Current Protocols (link below). There are a lot of helpful details and tips that didn't make the cut in the paper, so follow this space to learn more.
I hope to convince you that incubation time is a critical factor in getting quality reproducible staining, and that by going slower, you can not only get better data, but you can save money as well.
To kick things off today, let's have a look at how staining intensity varies even in short time frames. With high affinity antibodies, there’s a tendency to think that incubation time isn’t important because we can get some staining really quickly. This belief is reinforced by the way we view flow data on a log scale, so something like a two-fold change looks like nothing.
As a first example, here is CXCR5 staining on mouse splenocytes at 30, 45 or 60min at an optimized concentration of antibody. I think these are fairly typical incubation times. The MFI of the positive population is increasing in a linear fashion, so it’s unclear whether all of the CXCR5 molecules have been labeled.
It’s important to consider here that with shorter times, you have a higher risk of variability. For 30min, a 10% variation in time is only 3min, or less than the time for someone to randomly ask a question when your timer goes off. There’s also more risk with shorter incubation times that your samples won’t be at the desired incubation temperature for the same amount of time in each experiment. If the fridge is being opened and closed, or it’s a hot day, your samples are likely to be, on average, considerably warmer than they might be on a cool day working solo.
So I’ve shown you some short incubation times. What happens if we extend the incubation time even further? As we see below, by leaving the staining overnight, the stain index stabilizes, and remains constant across several hours. So we don’t have to worry about setting a precise timer anymore in order to get reproducible results.
Personally, I find this overnight staining method convenient. I can leave the staining, go home, and use the cytometer in the morning when it’s usually free. That’s a big plus right there. I’m also not running samples late at night when I’m tired and likely to make mistakes.
But, there’s something else that happens with overnight staining. Because if we leave the staining longer, the optimal dilution shifts. And shifts dramatically in many cases. In this example, the titration suggests we'd want to use the anti-CXCR5 at 1:500 rather than 1:50.
Obviously, this lower dilution isn’t something we’d want to use for a short staining, but with an overnight incubation, the staining is better separated than anything I can get with a short incubation, and ten times cheaper.
As I mentioned, this will be the first in the series, so stay tuned for more info about how to use overnight staining for better results with:
Reducing batch effects
Nuclear staining
Cytokine staining
Phospho-epitope staining
Reducing non-specific binding
High parameter panel design
Reagent info: https://www.thermofisher.com/antibody/product/CD185-CXCR5-Antibody-clone-SPRCL5-Monoclonal/61-7185-82