Midlands Innovation Flow Cytometry Conference 2026 resources

Kristina Tomkova, Jonathan W. Lewis, Thomas A. Nicholson, Thomas A. Jackson, Helen M. McGettrick, Jon Hazeldine.

Department of Inflammation and Ageing, University of Birmingham, UK.

Altered cellular metabolism is a hallmark of ageing and is thought to contribute to immunosenescence and sarcopenia. However, age-associated metabolic shifts within specific immune and stromal cell subsets in the circulation and peripheral tissues remain poorly characterised. Improved high-resolution profiling of cellular metabolism may reveal novel targets to mitigate age-related cellular dysfunction. In this study, we aimed to validate a combined approach integrating spectral flow cytometry with Single-Cell Energetic Metabolism by Profiling Translation Inhibition (SCENITH) to enable detailed metabolic assessment at single-cell resolution.

We developed a novel 30-colour spectral flow cytometry panel capable of simultaneously identifying multiple T-cell, B-cell, NK-cell, monocyte, stromal, and endothelial cell subsets within a single sample of 300K peripheral blood mononuclear cells (PBMCs). SCENITH – a flow cytometry–based technique – quantifies the cellular metabolic activity by measuring puromycin incorporation into newly synthesised proteins following targeted metabolic inhibition. Using four inhibitors targeting distinct metabolic pathways, we assessed glucose dependence, mitochondrial dependence, glycolytic capacity, and fatty acid and amino acid oxidation capacity at the single-cell level.

Method optimisation demonstrated that anticoagulant used to collect blood samples has an effect on the cellular metabolism. Blood collected in EDTA tubes had better preserved cellular metabolic responsiveness compered to Lithium-heparin tubes, as evidenced by increased sensitivity to oligomycin and 2-deoxyglucose treatment. Both fresh and cryopreserved PBMCs responded appropriately to mitochondrial inhibition, while cryopreserved cells showed enhanced glycolytic reliance. As a proof-of-concept, PBMCs from aged (n=3) and young (n=3) volunteers were analysed using the combined platform. Distinct metabolic signatures were observed across immune cell subsets between age groups, reflected by differential dependencies on glucose, mitochondrial respiration, and oxidative pathways. Although limited by small sample size, these findings demonstrate the feasibility and translational potential of this integrated approach for high-resolution metabolic profiling in ageing research.

Annabelle Bennett, Priyanka Chevour, Mohamed Hamed, Daniela Moreno Vicencio, Sascha Ott and Martin S. Davey.

Biomedical Science Directorate, Warwick Medical School, University of Warwick.

Colorectal cancer (CRC) is the second leading cause for cancer-associated mortality worldwide giving a vital urgency to find new immunotherapeutic targets. Unlike conventional ⍺β T-cells, γδ T-cells are a unique, unconventional lymphocyte subset that display both innate and adaptive characteristics. There are two major subsets of γδ T-cells; (1) innate-like V𝛿2+ γδ T-cells, primarily seen in blood, and (2) a population of tissue-associated V𝛿1+ γδ T-cells. Current understanding of γδ T-cells in CRC remains largely unknown, with both anti- and pro-tumour roles being uncovered. We hypothesise that clonally expanded tumour infiltrating γδ T-cells have distinct effector-like, tissue-resident and cytolytic phenotypes that may drive antigen-specific immune responses within the tumour microenvironment.

Methods

To investigate these phenotypes, we have developed a 38-colour spectral flow cytometry panel targeting both surface and intracellular proteins/peptides.  Using this panel, we analysed 18 matched blood, normal adjacent tissue (NAT) and tumour tissue samples from patients undergoing colorectal resection surgery.

Results

We find in blood that γδ T-cells, normally dominated by the V𝛿2+ T-cell subset, are often skewed towards CX3CR1+ CD45RA^hi V𝛿1+ T-cells, consistent with a shift towards an effector-like phenotype. Within matched NAT and Tumour tissues, we predominantly find CD103+CD69+NKp46+ tissue-associated V𝛿1+ T-cells. Moreover, Vδ1+ T-cells in NAT had elevated CTLA-4 expression, and tumour infiltrating tissue-associated Vδ1+ T-cells had elevated PD-1 and CD11a expression.

Conclusions

Together, colorectal tissue-associated V𝛿1+ γδ T-cells readily infiltrate tumours and are phenotypically similar to NAT-associated V𝛿1+ γδ T-cells. However, tumour infiltrating V𝛿1+ γδ T-cells are altered in their immune checkpoint expression and adhesion molecules, that may lead to immune regulation by the tumour. Determining targets to reactivate anti-tumour tissue-associated γδ T-cells could provide potential new immunotherapeutic targets for colorectal cancer.

Bradley Mason ¹, Laura Justham ¹, Liam Whitby ² Alison Whitby ², Stuart Scott ², Samuel Nti ², Jon Petzing ¹

¹ Wolfson School of Mechanical, Electrical & Manufacturing Engineering, Loughborough University, Loughborough, UK.

² UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospital NHS Foundation Trust, Sheffield, UK.

Modern flow cytometry (FC) is widely regarded as the de facto standard for high-throughput single-cell analysis in biological research, clinical and industrial settings. However, due to the intrinsic relative nature of empirical FC data, and the lack of definitive cell-by-cell cluster provenance associated with biological samples, users face significant challenges in validating the metrological precision and biological accuracy of FC data analysis. Effectuating challenges in the implementation of much needed quality assurance measures in FC analysis.

Our research involves the model-based generation of fully-controllable high-dimensional synthetic flow cytometry samples, which faithfully represent the characteristics of real-world data. All the while retaining the benefits of computational generation such as individual cell/event-level cluster lineage; contextual sample type/subtype adaptability and reproducible scalability of individual datasets.

In our work, we demonstrate that synthetic flow cytometry data can serve as both a verification tool to test and validate existing clustering methods, but also as a training and innovation tool to develop new approaches. For example, in manual clustering, quasi-identical samples quantify operator and software variability, providing absolute measures of gating uncertainty and bias across operators, training levels, laboratories and rare sample types. Benchmarking labelled synthetic data enables the evaluation of gating strategies or algorithmic clustering across differing sample subtypes. Furthermore, standards bodies, educators and developers can create synthetic ‘digital reference samples’ to support external quality assessment, refine standard operating procedures, and advance machine-learning applications.

In conclusion, labelled synthetic flow-cytometry datasets have a clear potential to improve diagnostic confidence in cytometry cluster analysis and tackle a critical challenge in regulatory-based quality assurance.

Emma Davey^[1], Omer Dushek^[2], John James^[1]

^[1] Warwick Medical School, University of Warwick.  ^[2] Sir William Dunn School of Pathology, University of Oxford.

Our immune system relies on T cells making appropriate and robust decisions to protect healthy cells whilst destroying pathogens or cancerous cells. To achieve this behaviour, extracellular cues from its receptors are converted into information through biochemical reactions within the cell that integrate to form a signalling network. Co-inhibitory receptors, such as Programmed Cell Death Protein 1 (PD-1), provide signals that suppress T cell activation and are of clinical interest as therapeutic targets for diseases such as cancer. Existing methods used to probe signalling networks lack the resolution needed to further the understanding of these complex systems, such as the decision-making processes used by T cells and the mechanism of action for co-inhibitory receptors.

We have therefore developed a toolkit of ratiometric fluorescent biosensors to probe the T cell signalling network of live cells using flow cytometry, capable of multiplexed measurements of multiple pathways simultaneously. By using these biosensors alongside novel optogenetic receptors, we can also precisely control T cell activation and co-inhibitory signals through illumination with blue light, whilst measuring clear signal outputs as well as achieving high temporal resolution.

This powerful, flow cytometry-based toolkit has enabled exploration of the dynamics of T cell activation signalling, as well as uncovering the differential impact of PD-1 on specific components of the signalling network. Future research will expand to study the impact of other co-inhibitory receptors, such as BTLA, TIGIT and LAG-3. We hope that creation of a detailed picture of T cell signalling dynamics through our novel methods will improve the understanding of these therapeutically important co-inhibitory receptors.

Megan Atherton-Frisby, John Pearl.

Division of Microbiology and Infection, College of Life Sciences, University of Leicester.

Pulmonary infection by non-tuberculous mycobacteria (NTM) is an increasingly serious problem afflicting those over 55 years of age and those in the agricultural sector. Patients with pulmonary NTM infections suffer from antibiotic treatment failure rates approaching 50% within 5 years with many patients developing life-long chronic lung disease.

We hypothesise that clinical treatment failure is, in part, caused by the robust and flexible respiratory capacity of NTM. To test this hypothesis, we used the fast-growing Mycolicibacterium smegmatis as a model to investigate the bioenergetics of single bacilli using flow cytometry. Flow cytometry is an excellent analytic platform as it provides single-bacteria resolution of membrane polarisation.

We report the development and use of an assay using the fluorescent dye 3,3′-Diethyloxacarbocyanine Iodide (DiOC₂(3)) as a differential reporter of bacterial respiratory capacity and overall health through the quantification of membrane polarisation, i.e., proton motive force. Validation of this assay included detection of bacterial-sized events by light scatter, fluorescent quantification of membrane polarisation in non-viable bacteria and modulation of membrane polarisation using specific respiratory inhibitors. Using this assay, we have identified the effects of different environmental growth conditions on membrane polarisation.

Based on these data, we conclude that DiOC₂(3) provides the basis of a simple flow cytometric assay for mycobacterial membrane polarisation which allows for sensitive and specific quantification of bacterial bioenergetics.

Sofina Iqra, Jill R. Johnson.

School of Biosciences, Aston University.

Pericytes are perivascular stromal cells involved in vascular stability, tissue repair, and fibrotic remodelling. However, reliable phenotypic identification of human placental pericytes using flow cytometry remains technically challenging due to marker overlap with mesenchymal stromal cells (MSCs), culture-induced variability, and sensitivity to experimental handling. The project focuses on the development and optimisation of a multi-parameter flow cytometry workflow to enable consistent characterisation of primary human placental pericytes in vitro. Panel design incorporates canonical pericyte-associated markers, including PDGFRβ and CD146, alongside exclusion markers to minimise endothelial contamination. Emphasis is placed on antibody titration strategy, fluorophore selection, compensation planning, and implementation of fluorescence-minus-one (FMO) controls to improve signal resolution and reproducibility. Optimisation parameters under evaluation include dissociation method, cell density at harvest, viability dye selection and gating hierarchy to reduce background fluorescence and improve discrimination of low-expression markers. In parallel, protocols are being established to assess phenotypic stability during in vitro expansion and following induction of mesenchymal lineage differentiation. Reactive oxygen species (ROS) detection using CellROX-based assays is being incorporated to examine potential interactions between oxidative stress and pericyte phenotype. This methodological framework aims to generate a reproducible cytometric pipeline for stromal cell phenotyping and to support future investigations into pericyte plasticity and their contribution to fibrotic tissue remodelling.

Adam Wright¹, Hnin Wint Wint Aung¹, Rebecca Dale¹, Abdullah Jamal¹, Tracy Thornton², Helen Evans², Phoebe Ashley², Sarah Edwards², Hamish McAuley^1,2, Omer Elneima^1,2, David J Cousins^1#, Neil Greening^1,2#, Chris E Brightling^1,2# (#co-last authors)

¹Institute for Lung Health, NIHR Leicester BRC Respiratory & Infection Theme, School of Medical Sciences, Division of Respiratory Sciences, University of Leicester, Leicester, UK

²University Hospitals of Leicester NHS Trust, Leicester, UK.

COPD Exacerbations are life threatening events and a significant proportion of these are characterised as ‘type-2’, occurring in the context of elevated blood and/or sputum eosinophils.  The source of type-2 cytokines such as IL-4, 5 and 13, during this time, are poorly defined. We examined cytokine (IL-4, 5, 9, 13, 17, IFNg, GMCSF, TNF) production from CRTh2/CD294⁺ blood CD8+ and CD4+ Type-2 (defined as TC2 or TH2, respectively) T cells at the single cell level, during stable and exacerbation clinic visits. Blood eosinophils were greater than 0.3 (x10⁹/L) and sputum eosinophils were > 3% of leukocytes during exacerbation.  Paired stable/exacerbation PBMCs (n=10) were stimulated with PMA/Ionomycin & Brefeldin A (5hrs). Intracellular cytokine staining was performed & analysed by cytometry.  Dimensionality reduction, clustering (X-shift) and cluster explorer were performed on a total of 114,000 events using FlowJo V10.10.  For each cluster, stable vs exacerbation comparisons were performed using a Wilcoxon signed rank test.

The total proportion of T2 cells were not different between stable and exacerbation visits. A total of 22 clusters were identified amongst T2 cells, spanning CD4⁺ (n=11), CD8⁺ (n=10) and CD4^–CD8^– (n=1) subsets; all were present during stable and exacerbation visits.  Six TC2 clusters were all significantly increased at exacerbation compared to stable state whereas only one TH2 cluster was differential (decreased).  Cumulative analysis of all 6 TC2 clusters showed that they were significantly increased at exacerbation vs stable (14±11 vs 9±6%, respectively, p=0.014) visits.  Phenotypically, these comprised cells producing 2+ (TNF, GM-CSF), 3+ (TNF, IL-13 with GMCSF or IL-4), 4+ (TNF, IL-13, IL-4 with GMCSF or IL-5) or all 5 cytokines. Cytokine production from CD4+ subsets, including IL-5⁺, were not increased.  T2-derived IL-17, IFN-g and IL-9 were not detected.  These data suggest that TC2 rather than TH2 cell frequency may be important during eosinophilic exacerbations in COPD.

Emma Jennings, Geroge Smith, Muna Fuyal, John James.

University of Warwick.

Background: Chimeric Antigen Receptor (CAR) T-cell therapy, involving the extraction of patient T-cells and arming them with receptors designed to target tumour specific antigens, has revolutionised the treatment of haematological malignancies in recent years. Despite the notable successes, CAR T-cell therapy only confers a durable response in 30-40 % of patients and exhibits a limited efficacy against solid tumours. Rapid exhaustion of CAR T-cells in response to persistent antigen stimulation is a known contributor to this limitation. Recently, T helper 9 (Th9) cells have been observed to have a more robust effectiveness against tumours, possessing an enhanced longevity.

Methods: Our work aims to explore the use of optically controllable pulsatile activation, thereby reducing the total CAR T-cell stimulation time, as a method of reducing cell exhaustion. Primary CD4+ T-cells are transduced with a photoactivatable anti-CD19 CAR construct. These OptoCAR T-cells are then co-cultured with CD19+ Raji B-cells under varying pulsatile sequences of light before flow cytometry was employed to explore their phenotype.

Results: Varied pulsatile signalling regimens led to altered expression of both co-inhibitory (e.g. PD-1) and co-stimulatory (e.g. ICOS) receptors on the surface of OptoCAR T-cells. We observed that pulsatile activation of OptoCAR T-cells was able to induce IL-9+ expression in the absence of TGF-β and IL-4 cytokine culture. Furthermore, a novel cytokine capture assay was developed and optimised to isolate live IL-9-secreting CAR+ T-cells for downstream functional assays and RNA sequencing.

Conclusion: Our data suggests that pulsatile activation warrants further investigation as a novel method for enhanced polarization of conventional CAR T-cells to a potentially more durable and effective therapeutic modality in the form of Th9 CAR T-cells.

Jessica Ting, Kamila Janaszek, Somaia Ghanem, Thomas Foley, Jill R. Johnson.

School of Biosciences, Aston University.

Background: Pericytes are multifunctional progenitor cells associated with the microvasculature, and have been increasingly recognised as key contributors to tissue fibrosis through their ability to transform into myofibroblasts under inflammatory conditions. These changes to pericyte phenotype include increased cell motility in migration assays, acquisition of an elongated spindle shape, and elevated expression of the myofibroblast marker a-smooth muscle actin (a-SMA) and the cell migration marker N-cadherin. Furthermore, we have shown in recent studies that chronic inflammation increases pericyte responsiveness to the chemokine CXCL12 via increased cell surface expression of CXCR4. However, the inflammatory mediators driving the acquisition of myofibroblast features by pericytes are yet to be elucidated.

Methods: We assessed the impact of fibrosis-associated inflammatory mediators (CTGF, bFGF, TGF-β1, and IL-13; 10 ng/mL for 7 days) on pericyte-to-myofibroblast transition in vitro. Immunostaining was performed to evaluate the expression of myofibroblast markers (a-SMA) and changes in cell shape, scratch assays were performed to assess the rate of cell migration, and flow cytometry was performed to determine cell surface expression of CXCR4 and N-cadherin as indicators of increased cell motility.

Results: We observed clear induction of a myofibroblast phenotype in human pericytes with all tested mediators. TGF-β1 and IL-13 were the most potent inducers, demonstrated by increased α-SMA expression (p<0.01), acquisition of a spindle shape (p<0.001), and accelerated cell migration (p<0.001). Using flow cytometry, we also observed that the MFI of CXCR4 and N-cadherin increased with these treatments (p<0.001). Conversely, CTGF only partially induced a myofibroblast phenotype in pericytes, with increased a-SMA expression (p<0.05) and accelerated scratch closure (P<0.01), but no appreciable increase in CXCR4 or N-cadherin by flow cytometry. bFGF induced a spindle shape (p<0.01), but decreased CXCR4 and N-cadherin expression (p<0.001).

Conclusions: These results demonstrate that IL-13 and TGF-b1 are potent drivers of pericyte-to-mesenchymal transition and additionally confer enhanced responsiveness to the inflammatory chemokine CXCL12.