Flow cytometry analyses the cells of a suspension one by one, at high throughput: several thousand — sometimes tens of thousands — of cells per second are interrogated for their size, granularity and expression of a panel of markers. It answers a recurring question in biology: what is this sample made of, and in what state are these cells? Where a bulk measurement drowns the signal in an average, cytometry restores the distribution cell by cell.
Principle and workflow
Cells in suspension are labelled with fluorochrome-conjugated antibodies, then carried one by one past one or more lasers. For each cell, the instrument records the scattered light (indicative of size and internal structure) and the fluorescence emitted by each marker. From this, through an analysis known as “gating”, subpopulations are defined by their marker combinations — for example activated CD3+ CD8+ T lymphocytes.
The rigour of an analysis owes as much to the bench as to the instrument: panel design (fluorochrome compatibility, marker hierarchy), antibody titration, compensation and “fluorescence-minus-one” controls to set positivity thresholds, and viability staining to exclude dead cells that would distort the results. These steps, often invisible in a publication, determine the reliability of the conclusions.
Variants and options
Several versions exist depending on the need. Conventional cytometry is enough for modest panels; spectral cytometry, which reads the full spectral fingerprint of each fluorochrome, allows panels of 30 markers and more, valuable for deep immunophenotyping. Cell sorting (FACS) does not merely analyse: it physically isolates the subpopulations of interest — for example antigen-specific B lymphocytes — to then submit them to fine molecular analyses such as single-cell sequencing. Beyond phenotyping, functional assays (proliferation, viability, cell cycle, intracellular cytokine production) turn the cytometer into a genuine bench for measuring cell state.
When and why this technique
Flow cytometry is used whenever cell populations must be quantified in a heterogeneous mixture, an immune response tracked, live cells sorted for a later step, or a functional parameter measured on a large number of cells. It is fast, quantitative and statistically robust.
The trade-off is clear. It loses spatial information: one knows that a cell expresses a marker, but not where it sat within the tissue — that is the domain of immunohistochemistry. It requires a cell suspension, hence a dissociation that may alter certain fragile epitopes. The number of simultaneous markers remains bounded by spectral overlaps (attenuated but not eliminated by spectral cytometry), and quality depends closely on sample viability. For an exhaustive transcriptional characterisation, single-cell sequencing takes over; cytometry then remains the upstream sorting step.
Inovarion’s expertise
Inovarion places flow cytometry at the heart of human immune-response analysis, designing panels and gating strategies as close as possible to each question. Its published work has used it to identify and FACS-sort antigen-specific B lymphocytes — for example the memory B cells directed against the SARS-CoV-2 Spike protein, detected using fluorescent antigen probes and dedicated gating strategies — upstream of their culture and single-cell sequencing. The same approach served to characterise the splenic B populations involved in immune thrombocytopenia relapses. Beyond haematology, the technique also enabled the phenotyping of non-immune populations, such as cultured human thymic epithelial cells. From panel design to sorting live cells, Inovarion tailors the strategy to the scientific objective.
See also: Single-cell RNA-seq (cell sorting upstream of sequencing).
Key publications
- Sokal et al. Maturation and persistence of the anti-SARS-CoV-2 memory B cell response. Cell, 2021. Record → · PubMed
- Crickx et al. Rituximab-resistant splenic memory B cells and newly engaged naive B cells fuel relapses in patients with immune thrombocytopenia. Science Translational Medicine, 2021. Record → · PubMed
- Villegas et al. Cultured Human Thymic-Derived Cells Display Medullary Thymic Epithelial Cell Phenotype and Functionality. Frontiers in Immunology, 2018. PubMed