Meet JESS: The latest addition to our biology capabilities

Meet JESS: The latest addition to our biology capabilities

We have recently purchased Jess, a fully automated western blotting device for our biology labs located in Cambridge, UK. Jess supports chemiluminescent and fluorescent detection up to picomolar level sensitivity with a built-in protein normalisation and highly reproducible results with molecular weight characterisation. It has broad applications for various biological targets, in multiple therapeutic areas.

While we are at it, let us also remind you of our current work in Immuno-oncology and cell cycle analysis using FACS. Please see below to learn more about our capabilities and how o2h discovery can provide insight-driven integrated drug discovery services across a wide range of modalities.


o2h scientist working with the newly installed JESS equipment at Hauxton House

About Jess

Jess is an automated platform by bio-techne for conducting protein western blotting under denaturing conditions using capillary fluidics. It has become an industry standard for providing detailed protein blotting data quickly, bypassing the lengthy and labour-intensive steps of traditional gel and membrane methods.

Functionality: The Jess system facilitates protein analysis, allowing characterization based on size, abundance, and post-translational modifications. It supports both fluorescent and chemiluminescent detection methods, along with technical enhancements like multiplexing, reprobing, and loading normalisation. ?

How Jess will benefit our clients:?We frequently support our clients in characterising proteins, which can be pivotal readouts on therapeutic target engagement, validation of mechanisms of action, or biomarkers of response. The samples providing these insights might be complex lysates from cell culture models, extracts from tissue samples, or even purified proteins. The speed with which these samples can be processed using Jess (hours, not days) and their reproducibility, thanks to the automation that runs well for multiplexing and screening roles at o2h discovery .


Immuno-oncology and cell population analysis

Overview:

Cancer immunotherapy, also known as immuno-oncology, is a form of cancer treatment that harnesses the power of the body’s own immune system to prevent, control, and eliminate cancer. The precise and dynamic nature of the immune system allows it to exclusively eliminate tumour cells and prevent cancer relapse. Immunotherapy has been an effective treatment for patients with certain types of cancer that have been resistant to chemotherapy and radiation treatment (e.g., melanoma). It holds the potential to become more precise, more personalised, and more effective than current cancer treatments—and potentially with fewer side effects.

Figure 1: Small molecule agents for cancer immunotherapy. Small molecules targeting pattern recognition receptors associated pathways, immune checkpoints, oncogenic signalling, metabolic pathways, cytokine/chemokine signalling, and immune-related kinases have been extensively investigated. Small-molecule immune modulators offer various advantages like oral bioavailability, short half-life, membrane permeability, extensive tissue penetration, low immunogenicity, and manageable adverse events, which lead to more potent antitumor activity and safer clinical application.?(Image source –
o2h scientist working with FACS CytoFLEX S equipment at India site

At o2h, we have developed a repository of healthy human donor derived peripheral blood mononuclear cells (PBMCs) which serve as starting material for various downstream immuno-oncology assays. These cells have been characterised by flow cytometry based immunophenotyping for multiple immune cell subsets using our in-house, flow cytometer CytoFLEX S, which comes equipped with 4 lasers and allows detection up to 15 channels.

Immunophenotyping enables expressed proteins on the surface of individual cells to be detected using fluorescently conjugated antibodies to define populations of interest. Cell subset identification may be combined with the analysis of activation status markers, homing markers or exhaustion markers. o2h team has experience in designing bespoke immunophenotyping panels, with the capability of detecting up to 8 colours simultaneously.

  • o2h scientists can utilise flow cytometry-based analysis to perform immune phenotyping of whole blood-derived PBMCs and quantify the expression of surface receptors and intracellular proteins.
  • Co-culture systems, such as PBMCs, T cells, and NK cells, can be characterised by their paracrine signalling, activation, proliferation, and differentiation using fluorochrome-conjugated antibodies against cell-surface receptors.
  • Therapeutic small molecules and antibodies can also 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 2. 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).

Cell Cycle Analysis

Assessing cell cycle distribution and proliferation is essential to study cell growth, differentiation, senescence and apoptosis. This helps to evaluate the underlying mechanisms, therapeutic efficacies of anticancer drugs and can be indicative of wider effects upon the cells, often used in the broader context or in conjunction with other target engagement assays.

Proliferating cells sequentially undergo transition through G1 - S - G2 and M phases, and under certain circumstances enter G0, as resting or quiescent state. ?During DNA damage, the progression of cell cycle is interrupted at certain checkpoints leading to failed chromosomal alignment and mitotic arrest or delays. These can be characterised at individual stages to exactly pinpoint disruption of the normal processes.

o2h scientists can implement flow cytometry-based analysis to profile mitotic pathways and evaluate your compounds for cancer therapies. Whole-cell staining with propidium iodide (PI) and antibodies towards specific proteins or surface markers helps to concurrently analyse cellular characteristics within distinct cell populations G0/G1, S, and G2/M. The software helps to quantitatively determine phase distribution along with characteristics like ploidy, apoptosis, senescence, cell type etc.

Read the complete case study here .

In Figure 3, we see the usual DNA profile of HeLa cells grown in culture. From this, we can divide the cell cycle into 3 main populations: G?/G? phase, S phase and G?/M phase based on the DNA content present within the cells.

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 G? 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 4: Separation of the G? 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 G? and M phases.

If you're interested in exploring our biology capabilities first hand, we’d be delighted to give you a tour of our Biology labs at Hauxton House. Meet our experienced team of biologists and discuss how we can support your drug discovery projects over a cup of masala chai or artisan coffee.

For more information or to schedule a visit, contact [email protected]


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