Flow cytometry is a powerful tool which enables the identification and characterization of individual cells (or other particles) within a population. Cells, beads or other analytes are flowed in a linear stream, individually interrogated by a series of lasers and measured through an array of detectors.
Individual cells can be characterized by their physical properties by analyzing how they deflect and defract the interrogating laser light.
Cell size and granularity can be determined by forward scatter (FSC) and side scatter (SSC) measurements, respectively.
Using just these properties it is possible to distinguish different cell types within a mixed population. A good example is the analysis of whole blood samples where lymphocytes, monocytes and granulocytes can be clearly distinguished without any markers necessary.
Figure 1: Identification of White Blood Cell Populations by Label Free Flow Cytometry Analysis
The multiplicity of flow cytometry readouts comes from the ability to excite fluorophores (from dyes or fluorophore tagged antibodies) on stained cells (or other particles).
Spatially separated lasers enable fluorophores to be excited across a range of wavelengths and the fluorescence emitted to be detected across a multitude of distinct detectors. For each event (cell, bead etc…) the combined physical and fluorescence parameters can then be used to identify the event and assess a plethora of phenotypic readouts.
Figure 2: Viable CD4+ T-cells within a peripheral blood mononuclear cell (PBMC) sample were identified and assayed for the proliferation marker CD25.
Incorporating antibodies to cell lineage markers allows individual cell types to be identified and analyzed within complex mixed populations. For example CD8+ and CD4+ T-cells can be identified (and analyzed) in a PBMC population using a combination of fluorophore conjugated anti- CD3, CD8 and CD4 antibodies.
In the same way protein expression levels can be assessed by the staining of cells with fluorophore conjugated antibodies to proteins of interest. This approach can be used for phenotypic readouts (e.g. γH2AX as a marker of DNA damage) and has been especially beneficial to PROTAC protein degradation projects.
In addition to antibody-based staining, many fluorescent dyes and probes are available to investigate phenotypic readouts. These include:
- viability dyes to measure cell health
- DNA binding dyes to monitor cell cycle progression
- Reagents to showcase apoptosis induction
Whilst historically under utilized in drug discovery, recent advances in cytometers with improved throughput and plate-based acquisition has enabled flow cytometry to provide robust, informative assays in drug discovery screening cascades.
With its acoustic focusing technology, flat top lasers and Cytkick max plate loader, the Attune Nxt allows us to utilize flow cytometry in many of our drug discovery projects.