Mass Cytometry vs Flow Cytometry

Mass cytometry, first introduced in 2009, is an advancement of flow cytometry that was developed to analyze more parameters per sample. Here, we look at how mass cytometry and fluorescence flow cytometry differ and offer guidance for method selection.

Fundamental differences

Both mass cytometry and flow cytometry are based on the concept of using labeled antibodies for multiplexed single-cell analysis. However, there are some fundamental differences between the two technologies. “Mass cytometry, also known as cytometry by time-of-flight (on which CyTOF^® systems are based), applies purified heavy-metal labels not normally found in biological systems,” explains Marla Lubinsky, Director, Marketing for Flow Cytometry and Imaging at Standard BioTools. “In contrast, flow cytometry relies on fluorophore-labeled antibodies for detecting cellular targets. This change in labeling enables mass cytometry to simultaneously detect 50 or more markers in the same sample, whereas flow cytometry is limited by the number of colors that can be distinguished without excessive overlap or autofluorescence.”

Currently, a typical flow cytometry experiment will detect around 8–10 different markers, although it is becoming easier for researchers to construct larger panels. “With the emergence of spectral flow cytometry and the optimization of high-dimensional flow cytometry, the number of parameters that can be measured in a single experiment is increasing,” reports Kenta Yamamoto, Product Manager, Cell Analysis at BioLegend. To illustrate this point, he highlights two recent publications describing the development of a 48 color mass cytometry panel  and a 43 color flow cytometry panel, and suggests that the gap between the two technologies is closing.

Beyond the types of labels that are used, the method of acquisition represents another distinction between mass cytometry and flow cytometry. “During mass cytometry analysis, the cells are atomized and ionized, then the metal tags are analyzed by time-of-flight mass spectrometry,” says Scott Weiss, Associate Director, Cytometry and CyTOF at CellCarta. “Flow cytometry acquisition instead involves running intact fluorescently tagged cells through a number of lasers and capturing the emitted light by detectors. In practical terms, while acquisition times are longer for mass cytometry due to lower cell throughput, higher parameter panels as well as barcoding technology can be used to augment this.”

Advantages and current limitations

So far, we have established that mass cytometry offers the advantage of being able to detect more markers per sample compared to flow cytometry, while flow cytometry has faster acquisition times and leaves the cells intact, meaning they can be retrieved for further use. But the two technologies each have several other benefits and limitations.

“Because a mass cytometer employs only one detector, it eliminates the need to perform special tuning or calibration for each experiment,” Lubinsky says. “Also, simultaneous analysis can be done from a single tube without requiring single-stained or autofluorescence controls, which represents an important advantage in situations where mouse and clinical research samples are in short supply. For example, using the Standard BioTools™ Maxpar® Direct™ Immune Profiling Assay™—a validated, dry-format antibody panel for use on the CyTOF XT™ and Helios™ mass cytometry systems—researchers can profile 30 immune markers from just 300 µL of blood, without having to use additional sample for staining controls.”

Another advantage of mass cytometry is that the metal tags are extremely stable and can withstand fixation/permeabilization and freezing without any change to the signal when the samples are processed. This makes it possible to stain cell surface and intracellular markers simultaneously and allows the labeled samples to be stored and later shipped for multi-site analyses. Fluorophores, on the other hand, are not resistant to the harsh workflows needed for phosphorylation markers or intracellular and functional targets.

On the flipside, a current limitation of mass cytometry relates to the fact that it is a relatively new technology compared to flow cytometry. “Commercially, heavy metal-conjugated antibodies are less widely available across vendors than fluorophore-conjugated antibodies,” says Yamamoto. “Therefore, if a large mass cytometry panel is desired, a significant portion of a researcher’s time may be dedicated toward self-reagent preparation, such as performing antibody conjugations in-house.” To simplify this process, BioLegend has developed Maxpar® Ready purified antibodies, which are optimized and ready to use with Standard BioTools’ Maxpar^® Antibody Labeling Kits.

The comparative infancy of mass cytometry also means that researchers who are used to a fluorescence flow cytometer might need additional training to operate a new instrument. “Currently, flow cytometry remains the go-to for smaller panels less than 18 colors, while mass cytometry tends to be deployed when greater than 20 markers are required,” notes Weiss. “But with both technologies continuing to push the limits of what can be measured in a single sample, preferences may change over time”.

Which technology to choose

When deciding between mass cytometry and flow cytometry, Weiss recommends that researchers begin by clarifying how many markers they need to detect. “If the number of markers is lower than 25, flow cytometry is most likely easier to deploy,” he says. “If above, mass cytometry is the more tested and truer platform for complex panels, although spectral flow cytometry is fast becoming an alternative option.” Yamamoto agrees, commenting that for most prospective users, flow cytometry is preferred for its high-throughput, user-friendliness, and instrument availability across institutes.

Lubinsky cautions against letting fluorescence limit your research question with a biased study design. “It is possible to bias your study by looking only at what you think might be important, rather than everything in a sample, based on the markers you have available,” she says. “Such an approach can result in missing cell populations or interactions you hadn’t expected, and can mean a novel signaling event or a significant effect on a certain cell type goes overlooked. CyTOF enables researchers to build a larger panel, providing not only granularity in cell populations of interest, but also outliers in one tube. This unique capability to capture diverse cell populations with granularity means targets not included in lower parameter fluorescent-based panels get captured with CyTOF routinely,” she adds.

With mass cytometry, you can more easily quantify the breadth and depth of the immune system, whereas flow cytometry is typically better suited to more basic—and smaller marker number—experiments.

Tips for experimental design

Whichever route you choose to go down, there are several steps you can take to ensure reliable results. “Because mass cytometry and flow cytometry begin by following very similar staining protocols, many best practices are applicable across both techniques,” says Yamamoto. “For example, when preparing your single-cell suspension, it is important that you implement measures to preserve target epitopes. And, for cell staining, using validated, high affinity antibodies is key. You will also want to understand the biology of your model system, including which markers are required for cellular identification and how abundantly those markers are expressed.”

When it comes to flow cytometry panel design, researchers can access a broad range of online tools, including Biocompare’s Flow Cytometry Panel Builder. For mass cytometry, Lubinsky says the validated Maxpar Direct Immune Profiling Assay is a good place to start. “Researchers can also capture other markers of interest by choosing from over 20 ready-to-go panels and a catalog of over 800 antibodies,” she says. And consider barcoding samples, a key to reducing technical variation and an easy step when using CyTOF technology.