Flow cytometry is a powerful and versatile tool widely used in cancer biology, immuno-oncology, cell cycle analysis, and mechanism of action studies. It enables rapid, multi-parametric analysis of cells in solution by utilizing lasers to produce scattered and fluorescent light signals. These signals are detected by photodiodes or photomultiplier tubes, allowing analysis of cell populations based on their fluorescent or light-scattering characteristics.

At o2h, we offer a comprehensive range of flow cytometry-based research services using the advanced CytoFLEX S from Beckman Coulter, to cater to the diverse needs of researchers. The state-of-the-art system supports up to 13 fluorescent channels, along with forward and side scatter, enabling detailed multi-parameter analysis. The system provides the following features:

• High sensitivity & resolution with APD detectors ensuring precise low-abundance signal detection for complex analyses
• Customizable configuration tailored with options to add or remove lasers and detectors, for various research needs
• User-friendly software CytExpert, streamlining data acquisition, analysis and reporting

o2h offers a wide range of flow cytometry services including but not limited to Cell cycle analysis, population analysis, health and viability, rare cells detection and cytokine analysis.

Cell cycle analysis

The cell cycle can be divided into 5 main stages: G₀ phase where cells are resting and not undergoing any division, G₁ phase where cells are preparing to undergo DNA replication, S phase where cells are actively replicating their DNA, G₂ phase where cells have completed DNA replication and M phase where cells are undergoing mitosis to split into 2 daughter cells.

At o2h, we have developed flow cytometry-based studies which allow the cell cycle phases to be dissected and detect any alterations that result from treatment.

Figure 1: Cell cycle stages of HeLa cells in exponential growth phase in culture determined by staining of DNA content.

These stages can also be manipulated in culture to trap cells in one part of the cell cycle or to enrich for certain populations. Figure 2 shows the effects of a double thymidine block on the progression of cells through the cell cycle. As can be seen, this traps the majority of cells into the G₀/G₁ phase before releasing the cells allows them to progress through the cell cycle.

Figure 2: The effects of a thymidine block on cell cycle progression in HeLa cells. Left panel shows cells which have undergone a double thymidine block, the majority of cells are trapped in the early G₀/G₁ phases of the cycle. The middle panel shows the cells 2 hours after the release of the block where the majority of cells are in S phase. The right panel shows the cell cycle stage 8 hours after release where most of the cells have progressed into the G₂/M phase of the cell cycle.

To gain additional insight into the stages of the cell cycle, it is possible to combine DNA content staining with other markers which can separate cell cycle stages, such as Phospho-Histone H3 to distinguish the G₂ and M phases. As can be seen in figure 3, a relatively small proportion of cells are in M phase actively undergoing mitosis, and this can be enriched by using nocodazole, an agent that traps cells in the mitotic phase. Being able to detect cells in the M phased can be used to investigate the rate of cell division which is linked to a range of therapeutic opportunities.

Figure 3: Separation of the G₂ and M phases of the cell cycle by phosphor-Histone H3 and PI staining. HeLa cells growing in culture treated with vehicle (right 2 panels) or nocodazole (left 2 panels) and co-stained for DNA content and phosphor-histone H3 (Ser10). This shows that nocodazole traps cells in the M phase and that pH3 can be used to separate the G₂ and M phases.

At o2h, we are experts in cellular and biochemical assays, with the ability to combine multiple study types such as cell cycle analysis, DNA ploidy with other assays including cell death and phenotypic screening, thus providing you with layers of analysis depth in a bespoke manner to fit your research needs

Cell population analysis

o2h offers services for the phenotypic screening of novel immune therapeutics and determination of the molecular mechanism of action of new anti-cancer immune modulators.

We have developed a repository of healthy human donor derived peripheral blood mononuclear cells (PBMCs) which serves as starting material for various downstream immuno-oncology assays. These cells have been characterized by flow cytometry based immunophenotyping for multiple immune cell subsets.

• o2h scientists can perform immune phenotyping of whole blood-derived PBMCs and quantify the expression of surface receptors and intracellular proteins
• Develop co-culture systems of various cancer cell lines with PBMCs, T cells and NK cells; characterize paracrine signalling events; activation, proliferation, and differentiation using fluorochrome-conjugated antibodies towards cell-surface receptors
• Therapeutic small molecules and antibodies can be screened for their cytotoxic potential against cancer cell lines using Annexin V/7-AAD dyes to quantify the percentage of cellular apoptosis and necrosis

Figure 4. Human PBMCs stained with a T cell antibody panel. The gating strategy involved first identifying the lymphocyte population by forward scatter (FSC) and side scatter (SSC). Live T cells (CD3+) were gated and then the two main types of T cells were defined by CD4+ (T helper cells) and CD8+ (cytotoxic T cells).

To know more about our biology capabilities and our broader services, please reach out to us at discovery@o2h.com.