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CyTOF (cytometry by time of flight) is mass cytometry: cells are stained with antibodies tagged with stable metal isotopes instead of fluorescent dyes, then atomized and ionized so a time-of-flight mass analyzer can count each isotope, one cell at a time. Because isotopes are told apart by mass rather than colour, the published reviews describe more than 40 measured parameters per cell without fluorescence compensation. The price is that the cells are destroyed in the measurement and the instrument is slower and costlier than conventional flow cytometry.
This page is written for lab staff and core-facility users deciding whether a mass cytometry experiment fits their question. It is general information, not a protocol, and it does not describe any one manufacturer’s current instrument.
Definition and scope
CyTOF is the name most often used for mass cytometry, a single-cell technique in which antibodies are labelled with heavy-metal isotopes rather than fluorophores. A 2026 narrative review puts it this way: mass cytometry "replaces fluorescent tags with heavy metal isotopes bound to antibodies," and a time-of-flight mass spectrometer detects the isotopes by mass, "removing spectral overlap" (Susuki et al., 2026). The term covers measurement of cells in suspension. Measuring cells in intact tissue sections is a related but separate method, imaging mass cytometry (see below).
How mass cytometry works
The workflow below follows a 2015 review of the platform (Arthritis Research & Therapy 2015;17:127), which describes the instrument generation of that time.
- Staining. Cells are labelled with antibodies tagged with stable metal isotopes. These isotopes are scarce in biological samples, which keeps background low.
- Nebulization. The cell suspension is nebulized into single cell-containing droplets, which are dried by heat.
- Atomization and ionization. The droplets enter an inductively coupled argon plasma, where each cell is atomized and ionized.
- Detection. The resulting ion cloud is measured by a time-of-flight (TOF) mass analyzer. The signal intensity at each isotope mass corresponds to the amount of one specific antibody on that cell.
Mass cytometry compared with fluorescence flow cytometry
Each row below traces to one of the two sources listed at the end. Throughput and parameter numbers come from the 2015 and 2026 reviews and are not specifications for any current model.
| Feature | Fluorescence flow cytometry | Mass cytometry (CyTOF) |
|---|---|---|
| Label detected | Fluorescent tags | Heavy-metal isotopes, detected by mass (2026 review) |
| Signal overlap between channels | Spectral overlap, handled by compensation | Removed by mass separation; no compensation across channels needed (2015 and 2026 reviews) |
| Parameters per cell | Varies by instrument | Up to 40 (2015); more than 40 (2026) |
| Forward and side scatter | Available | Not available (2015) |
| Tag sensitivity | PE and APC are more sensitive | Metal tags are less sensitive than PE or APC (2015) |
| Speed | Faster | Roughly 500 events per second (2015) |
| Cells after measurement | Not stated as a limit in the 2015 review | Destroyed; cannot be recovered for functional or transcriptional work (2015) |
| Tissue location | Not retained for dissociated cells | Conventional CyTOF cannot show where cells sat in tissue (2026) |
Related techniques
Imaging mass cytometry
The 2026 review states that imaging mass cytometry (IMC) can preserve spatial information and complement CyTOF data from dissociated cells. For the wider landscape of tissue-mapping methods, see spatial proteomics methods for tissue mapping.
Conventional and spectral flow cytometry
For how fluorescence instruments work and how their plots are read, see flow cytometry: how it works. The spectral flow cytometry guide covers the fluorescence-based approach to high-parameter panels, and imaging versus conventional flow cytometry covers another variant.
Where it matters for labs and core facilities
A checklist drawn from the limits listed in the two reviews, for planning a mass cytometry experiment:
- Sample consumption. Cells are atomized, so there is no sorting or re-analysis of the same cells afterwards. The 2015 review also notes that roughly two thirds of cells ejected from the nebulizer do not reach the detector as ion clouds, so input numbers should be discussed with the instrument operator.
- Single-cell suspension. The 2026 review notes that some tissues are difficult to convert into single-cell suspensions, which limits what can be run.
- No scatter parameters. Size and granularity readouts from scatter are not available, so panels must identify cells by their markers.
- Cost and access. Both reviews cite higher costs and specialized instrumentation, which is why such instruments are typically a shared resource. See core facilities for how shared research cores are organized.
Common misunderstandings
- “CyTOF is a fluorescence method.” It is not; detection is by mass of metal isotopes.
- “The cells can be recovered afterwards.” They cannot; they are atomized and ionized in the plasma.
- “It is just faster flow.” It is slower, at roughly 500 events per second in the 2015 review.
- “It shows where cells are in tissue.” Conventional CyTOF does not; imaging mass cytometry addresses that.
FAQ
What does CyTOF stand for?
Cytometry by time of flight. It refers to the time-of-flight mass analyzer used to detect metal-tagged antibodies, and is used interchangeably with mass cytometry.
How is CyTOF different from flow cytometry?
It detects metal isotope tags by mass instead of fluorescent tags by light, which removes spectral overlap and allows more than 40 parameters per cell, but it has no scatter signals, is slower and destroys the cells (see the table above).
Can cells be recovered after CyTOF?
No. Cells are atomized and ionized during measurement, so they cannot be used for downstream functional or transcriptional analysis.
Sources
- Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy. Arthritis Research & Therapy 2015;17:127. doi:10.1186/s13075-015-0644-z. pmc.ncbi.nlm.nih.gov/articles/PMC4436107 (read 2026-11-08).
- Susuki Y et al. Immune profiling in orthopaedic diseases using mass cytometry: a narrative review. Bone & Joint Research 2026;15(5):497-507. doi:10.1302/2046-3758.155.BJR-2025-0548.R1. pmc.ncbi.nlm.nih.gov/articles/PMC13169456 (read 2026-11-08).
