Spectral Flow Cytometry: Separating the Fluorophore Rainbow

Flow cytometry is a widely used analytical technique that distinguishes between cell populations depending on the presence or absence of chosen markers. However, the number of markers that can be included in a flow cytometry panel is limited by the number of fluorophores with distinguishable emission spectra. Spectral flow cytometry, an evolved iteration of conventional flow cytometry, overcomes this problem by differentiating between similarly emitting fluorophores, thereby increasing panel sizes.

Kaitlyn Sadtler, an Earl Stadtman Investigator and Chief of the Section for Immunoengineering at the National Institute of Biomedical Imaging and Bioengineering (NIBIB), studies the immune response to tissue trauma and medical device implantation. In a recently published Cell Tissue Organs paper, Sadtler’s laboratory developed a 24-color, spectral flow cytometry panel to investigate immune cell infiltration at the site of muscle injury within rats.¹

In an interview with The Scientist, Sadtler discussed why and how she developed her spectral flow cytometry panel for rats, as well the advantages and disadvantages of spectral flow cytometry.

What is flow cytometry and spectral flow cytometry?

Flow cytometry measures the expression of different proteins on cells one at a time while they are flowing through a machine. You can think of it as marbles through a garden hose. As each marble passes through, there are detectors to say, “this marble has orange in it” or “this marble has purple in it.” Those different colors represent the fluorophores’ peak emissions and this tells us what proteins are present on the cells.

Everything in life and science is messy. Some fluorophores emit over a variety of wavelengths, so some marbles have red-orange inside them as opposed to just orange or red. Spectral flow cytometry is a take on flow cytometry, which instead of looking at a peak in a specific channel (i.e., the orange channel or the red channel), it looks at the pattern of the whole spectra. This technique allows researchers to tease apart signals from very similarly colored fluorophores.

Why did you generate a spectral flow cytometry panel for rats?

We work at the intersection of immunology and bioengineering, where each field has a different model system. Mice have been the workhorse for immunology for a very long time and as a result there is an absolute plethora of reagents. However, researchers use rats more frequently when evaluating medical devices and biomaterials, but reagent availability is limited. We wanted to bridge the gap between basic, mechanistic immunology studies that happen in mouse models versus the biomaterial studies that happen in rat models. Our goal was to figure out what tools are out there for rats, assemble them into a usable toolbox, and present said toolbox. The spectral flow cytometer allowed us to use similarly colored fluorophores that could not necessarily be used together on a standard flow cytometer.

How did you construct your panel for spectral flow cytometry?

To construct our panel, Kenneth Adusei, the lead author of the paper, and I performed a product search of the various rat antibodies that were available off-the-shelf. We created a reagent spreadsheet that stated which vendors had which antibodies and in which colors. As for choosing which fluorophores to use, we needed to evaluate the autofluorescence of our samples to avoid the peak channels where autofluorescence was coming in.

What are the advantages and disadvantages of spectral flow cytometry?

Spectral flow cytometry allows scientists to distinguish between the real signal of a fluorophore versus what signal might be bleeding through from an overlapping fluorophore. This lets researchers measure more colors at a time with confidence. They get more of the information they want, but also noise from autofluorescence. We work with highly autofluorescent samples, and some of this autofluorescence is from the myeloid cells themselves, which shine bright with pretty colors before we stain them. This gives us an extra variable to deal with. But autofluorescence provides a lot of information as well. For example, we can identify eosinophils by their autofluorescence alone, without any markers. This points to the future recognition of different cell populations based off their autofluorescence. Though the extra noise is a pain, that noise has information within it. We just have to read through the signal.

This interview has been condensed and edited for clarity.