Getting there
Holywell Park Conference Centre, 25th April 2022
Loughborough University Science and Enterprise Park, Holywell Way, Loughborough LE11 3GR. By Car: there are plenty of free onsite parking By Public Transport: Buses depart from Loughborough train station every 10 minutes (Kinchbus Sprint Timetable) taking 20minutes (15 minute taxi ride). Contact
Contact David Onion.
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Mini ProgrammeMI Flow Cytometry Meeting
Holywell Park, Loughborough University, 25th April, 2022 9:00 Registration 9:30 Introduction to the Midlands Innovation Flow Cytometry Group 9:45 Session 1: - Academic Session 10:30 Coffee break 11:00 Session 2: - Academic Session 11:50 Workshops 1 12:30 Lunch, Posters and Exhibition 13:30 Session 3: – Academic Session 14:15 Workshop 2 15:15 Break 15:30 Keynote speaker 16:15 Closing Remarks and Prizes 16:30 Meeting Close and Drinks |
Thanks to our Sponsors
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Full Programme
Programme
9:00 Registration (Babbage Room)
9:30 Welcome (Turing Room)
Introduction to the Midlands Innovation Flow Cytometry Group
9:45 Session 1 (Turing Room)
Jon Petzing (Loughborough University) - Flow cytometry applied to cell analysis and therapies. Why is there variation in the metrology?
Georgina Hopkins (University of Nottingham) - Development of an in vitro, human, cell-based assay to investigate the role of lipids and invariant NKT cells in allergic sensitisation.
Matthew Roberts (Loughborough University) - Physical activity is associated with reduced tethering and migration of pro-inflammatory monocytes in White European and South Asian males with and without central obesity.
10:30 Coffee Break (Babbage Room)
11:00 Session 2 (Turing Room)
Rebecca Drummond (University of Birmingham) - Tracking host-fungal interactions in vivo.
Nancy Gomez (University of Nottingham) - The effect of cigarette smoke on the production of extracellular vesicles by immune cells.
Jaspreet Bansal (Aston University) - Developing a flow cytometry-based toolkit for assessing mesenchymal stromal cell functional properties.
11:50 Workshop 1
Beginners (Murdoch/Brunel Room)
Lindsay Bentley (Miltenyi Biotech) - Improving flow analysis through sample prep.
Stephen Rackstraw (ThermoFisher) – Why are researchers excited about spectral flow cytometry?
Advanced (Turing Room)
Vendula Sinkorova (SONY) – Spectral Cell Analysis and its advantages.
Alice Law (NanoFCM) – NanoAnalyzer: Exploring the nano-sized with a dedicated flow cytometry platform.
Cell Sorting Demonstration (Kelvin Room)
Ian Brotherick (Beckman Coulter) – Cell sorting demonstration with the CytoFLEX SRT.
12:30 Lunch, Posters and Exhibition (Babbage Room)
13:30 Session 3 (Turing Room)
Jill Johnson (Aston University) - CXCL12 drives pericyte accumulation and airway remodelling in allergic airway disease.
Andrea White (University of Birmingham) - CXCL12 Identifies heterogeneity in cortical epithelial cells.
Rasha Kahder (University of Nottingham) - Protein Kinase C-δ C2 domain interactions in breast cancer
14:15 Workshop 2
Beginners (Murdoch/Brunel Room)
Mike Blundell (Biorad) - How to avoid common pitfalls to get publication quality flow cytometry data.
Adam Davison (Cytek) – Full Spectrum Profiling™ (FSP) – Best practices in reference control selection, optimisation and longitudinal performance validation.
Idhnan Hussain (CURIOX Biosystems) - Next-Generation cell preparation with laminar wash technology.
Advanced (Turing Room)
Thomas Adejumo (Fluidigm) - How to set-up and run high parameter cytometry experiments.
Pj Chana - (Luminex) Join the revolution… Demonstrating how AI can simplify & enhance complex Imaging Flow Cytometry analysis.
Morgan Blaylock (BD Biosciences) - Road to Complex Biology: streamlining the power of high-dimensional biology with BD CellView™ and BD Rhapsody™.
Cell Sorting Demonstration (Kelvin Room)
Ian Brotherick (Beckman Coulter) – Cell sorting demonstration with the CytoFLEX SRT.
15:15 Break (Babbage Room)
15:30 Keynote Speaker (Turing Room)
Professor Andrew MacDonald (The University of Manchester) – Dendritic cells and macrophages in promotion and regulation of pulmonary type 2 inflammation.
16:15 Concluding Remarks and Prizes (Turing Room)
16:30 Meeting Close & Drinks
Oral Presentations
Session 1
Flow Cytometry Applied To Cell Analysis And Therapies –Why Is There Variation In The Metrology?
Jon Petzing, Centre for Biological Engineering, Wolfson School of Mechanical, Electrical & Manufacturing Engineering, Loughborough University. j.petzing@lboro.ac.uk
Cell analysis and production of cell therapies, whether by single clinical or multiple biomanufacturing sites, introduces challenges to be solved to achieve the delivery of safe, viable diagnosis and therapeutics. From a biomanufacturing viewpoint, these challenges include the translation of the process from T-flask to bioreactor, inline measurement and assessment of the cell journey, concerns around infections and asepsis, correct cell differentiation, supply logistics, business considerations, and the quality and variation of starting materials. This latter component was investigated and considered in the context of Haematopoietic Stem Cells Therapy (HSCT) with orders of magnitude variation found within the literature. A single site clinical centre HSCT exemplar provided opportunity to further investigate the associated HSCT cell manipulation processes. It was observed that the Flow Cytometry processes using operator defined manual gating techniques was potentially prone to variation in data analysis as a function of changing operators. Bespoke studies were subsequently commissioned with 30+ operators being asked to manually gate and process increasingly complex cell data. The results demonstrated that as complexity increased then cell count data variation increased, but, if systematic protocols were put into place then this human operator variation could be mitigated.
Whilst manual gating is (in many cases) the preferred method for flow cytometry data processing, it was clear that a proliferation of unsupervised automated software platforms were being made increasingly available and competing for the data processing task. The aim of many of these software solutions is to reduce operator variation and increase the repeatability and accuracy of cell enumeration. Yet these software solutions involve a range of different mathematical paradigms that in themselves offer the potential for introducing different final cell count values and hence variation into the clinical and biomanufacturing setting. The research completed here has been to develop agnostic synthetic two and three cluster data sets that allows bespoke testing of a range of software platforms. This offers opportunity for unambiguous statements of accuracy and repeatability for each software solution, thus leading to a comparability analysis.
This presentation considers how variation in the metrology of flow cytometry (specifically the data analysis component) can potentially impact quality of diagnosis within the clinical setting, and also affect the decision making associated with cell therapy critical quality attributes from a biomanufacturing perspective.
Development of an In Vitro, Human, Cell-based Assay to Investigate the Role of Lipids and Invariant NKT cells in Allergic Sensitisation
Georgina V Hopkins1, Stella Cochrane2, David Onion1, and Lucy C Fairclough, 1
1School of Life Sciences, The University of Nottingham, Nottingham NG7 2UH, UK
2SEAC, Unilever, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ, UK
Abstract
Background
Immunoglobulin E (IgE)-mediated allergies are increasing in prevalence. However, the mechanisms underpinning the first phase of IgE-mediated allergy, allergic sensitisation, are still not clear. Recently, the potential involvement of lipids in allergic sensitisation has been proposed, with reports that they can bind allergenic proteins and act on immune cells to skew to a T helper type 2 (Th2) response. However, the existing research in this area is limited and predominantly uses murine models.
Objectives
The aim of this research it to develop an in vitro method to study the role of lipids and invariant natural killer T (iNKT) cells utilising a human model of allergic sensitisation.
Methods
Due to low abundance of iNKT cells in human peripheral blood, iNKT cells were expanded over 14 days by stimulation with the glycolipid, α-GalCer, which is the most potent activator of iNKT cells. The iNKT cells were then isolated and co-cultured with autologous, α-GalCer-pulsed dendritic cells (DCs) to allow iNKT-lipid binding. Th1 and Th2 iNKT cell cytokine release were then measured by flow cytometry. The internalisation of α-GalCer by DCs was confirmed by imaging cytometry.
Results
The results indicated α-GalCer induced an 80-fold expansion of iNKT cells by Day 14. Expansion with α-GalCer also induced a shift in iNKT cell phenotype from predominantly CD4-CD8- at Day 0, to predominantly CD4+CD8- iNKT cells by Day 14. The iNKTs co-cultured with autologous α-GalCer-pulsed DCs displayed increased IL-4 and IFN-y secretion within 5 hours of co-culture. Fluorescent α-GalCer was successfully internalised by immature DCs.
Conclusion
Overall, this establishes a human model system where allergen-associated lipids, such as peanut lipids, can be used in substitute of α-GalCer, to determine whether lipids enhance iNKT cell Th2 cytokine secretion, shifting towards a state of allergic sensitisation.
Physical activity is associated with reduced tethering and migration of pro-inflammatory monocytes in White European and South Asian males with and without central obesity.
Matthew J Roberts1,2, Malik Hamrouni1,2, Alex J Wadley3, Nicolette C Bishop1,2.
1 National Centre for Sport and Exercise Medicine, School of Sport, Exercise and Health Sciences, Loughborough University, Epinal Way, Loughborough, United Kingdom, LE11 3TU.
2 National Institute for Health Research (NIHR) Leicester Biomedical Research Centre, University Hospitals of Leicester, National Health Service (NHS) Trust and the University of Leicester, Leicester, United Kingdom, LE1 5WW.
3 School of Sport, Exercise & Rehabilitation Sciences, College of Life & Environmental Sciences, University of Birmingham, B15 2TT.
Migration of monocytes from the circulation into adipose tissue is a key event in developing chronic systemic inflammation in obesity and is related to increased cardiometabolic disease risk. Using an ex vivo model to mimic blood flow dynamics and endothelial integrin expression - coupled with flow cytometry - we previously reported monocyte adherence and migration is higher in men with central obesity versus lean men. Importantly, higher physical activity mitigated the effect of obesity on cell movement, independent of weight loss. We now wanted to investigate if this effect was influenced by ethnicity, as South Asians (SAs) have a higher risk for cardiometabolic disease than White Europeans (WEs).
Using the same ex vivo model (Figure 1) in SA and WE men with and without central obesity (N=10 in each group), we collected 1.75 x 105 PBMCs before migration (baseline) and tethered and migrated PBMCs migrating towards an adipose-tissue conditioned media. CD14 and CD16 expression (monocyte subsets) and chemokine receptor expression (CCR2, CCR5) was determined using four-colour flow cytometry.
Our preliminary evidence suggests monocyte tethering and migration towards adipose tissue are exacerbated in SA men with central obesity versus SA men who are lean and versus WE men with and without central obesity, independent of circulating monocyte numbers. Significantly, our data suggest that higher physical activity can mitigate the effect of obesity and ethnicity on cell movement. This is encouraging given the increased cardiometabolic disease risk, low levels of physical activity, and high levels of central adiposity in SAs.
Session 2
Tracking Host-Fungal Interactions In Vivo
Rebecca Drummond. Institute of Immunology & Immunotherapy / Institute of Microbiology & Infection, University of Birmingham.
Fungal infections of the central nervous system (CNS) are often life-threatening and typically affect patients with AIDS, CARD9 deficiency or iatrogenic immunosuppression. The most common cause of human fungal brain infection is Cryptococcus neoformans, which causes over 150,000 deaths each year. Our laboratory tries to understand how resident phagocytes within the brain, called microglia, recognise and respond to invading Cryptococcus fungi. We use fungal strains that express fluorescent proteins to track their interactions with host microglia, and use multi-colour flow cytometry to analyse the quantity and quality of such interactions in the brain. Using these approaches, we have found that microglia become hosts for intracellular fungal growth and that this interaction promotes brain fungal infection. We hypothesised that the fungus utilises microglia to gain access to restricted nutrients in the brain, such as copper. To test that, we generated a fluorescent-reporter fungal strain that switches on GFP expression when importing copper. Using flow cytometry, we found that GFP expression is higher in fungal cells that are bound within microglia, compared to ‘free’ fungal cells that are not associated with host cells. Therefore, Cryptococcus fungi upregulate copper importing machinery primarily within microglia, which explains the fungal dependence on microglia for early growth in the brain.
The effect of cigarette smoke on the production of extracellular vesicles by immune cells
Nancy Gomez1, David Onion1, Victoria James2 and Lucy Fairclough1
1School of Life Sciences, The University of Nottingham. 2School of Veterinary Medicine, The University of Nottingham
Smoking or other inhaled irritants induce an inflammatory response which plays a critical role in the initiation and progression of Chronic Obstructive Pulmonary Disease (COPD). Extracellular vesicles (EVs) originating from the airways have the potential to initiate and propagate inflammation in lung and systemic inflammatory conditions. The aim of this study is to examine the effect of cigarette smoke on the production of extracellular vesicles by cells of the immune system. Isolated peripheral blood mononuclear cells (PBMCs) were exposed to cigarette smoke extract (CSE) for 24 hours. After defining the presence of EVs by transmission electron microscopy (TEM), Calcein-AM and fluorophore-associated anti-tetraspanin monoclonal antibodies (moAbs) for CD9, CD63 and CD81 were added, and EVs/mL were measured by imaging flow cytometry (IFC). PBMC-derived EVs were isolated by size exclusion chromatography and were profiled using the MACSPlex exosome kit. The presence of EVs was confirmed by TEM. EV numbers and tetraspanins expression were identified by IFC, showing that EVs/mL increased with exposure to CSE and that addition of multiple anti-tetraspanin moAbs does not affect detection of any tetraspanin. Key cell markers on EV membranes for each CSE concentration were identified. This study demonstrates increased EVs release from PBMCs following exposure to CSE and shows the multiple tetraspanins and membrane markers on EVs. Future work will observe the effect of EV uptake on T cell phenotype.
Developing a flow cytometry based toolkit for assessing mesenchymal stromal cell functional properties
Jaspreet Bansal – Aston University
Mesenchymal stromal cells (MSC) are non-hematopoietic tissue resident progenitor cells and are a key mediator for cell based therapies. MSCs have low immunogenicity and crucially can both promote resolution of inflammatory conditions through modulation of immune cells as well as promoting tissue repair. However, MSCs are a rare population of cells in the body and require to be expanded ex vivo to provide the large number of cells required for therapy. Unfortunately, during expansion in culture, MSCs spontaneously differentiate, losing their clinically important repair and immuno-modulatory properties, instead become lineage committed stromal cells. Currently we cannot define clinically efficacious naïve multipotent, immuno-modulatory MSCs from their more mature progeny using phenotypic surface markers. Therefore, different forms of functional analysis have to be employed to determine the potential of laboratory expanded MSCs.
We have developed a flow cytometry based toolkit of functional measurements to assess changes in MSC physiology. This allows monitoring of distinct physiological processes known to correlate to MSC immuno-modulatory potential. Our workflow uses a toolkit to determine changes in metabolism (mitochondrial activity), senescence (b-Galactosidase activity, cell surface phenotype as well as the measuring the MSCs immuno-modulatory potential with immune cells (T cell suppression, induction of Tregs). We propose this flow cytometry based approach allows evaluation of multiple processes known to impact on MSC activity and is a useful strategy for measuring the effects of different culture conditions on MSC function, and how this may impact on their subsequent use for patients.
Session 3
CXCL12 drives pericyte accumulation and airway remodeling in allergic airway disease
Jill Johnson, College of Health and Life Sciences, Aston University.
Background: Airway remodeling is a significant contributor to impaired lung function in chronic allergic airway disease, and pulmonary pericytes have been shown to significantly contribute to airway wall remodeling and lung dysfunction in a mouse model of asthma. This study aimed to characterize pulmonary pericytes in healthy and inflamed lungs, and to elucidate the mechanism by which pericytes accumulate in the airway wall in a mouse model of chronic allergic airway inflammation driven by respiratory exposure to house dust mite (HDM) extract.
Methods: Magnetic sorting for CD146+ pericytes was performed on healthy mice, and these cells were then stained for cell surface markers using the Biolegend LegendScreenTM and assessed by flow cytometry. Subsequently, mice were subjected to a protocol of chronic airway inflammation driven by intranasal delivery of the common environmental aeroallergen HDM. Phenotypic changes to lung pericytes were assessed by flow cytometry and immunostaining, and the functional capacity of these cells was evaluated using in vitro migration assays. The molecular mechanisms driving these processes were targeted pharmacologically in vivo and in vitro using the CXCL12 neutraligand LIT-927.
Results The LegendScreenTM indicated the absence of hematopoietic cell markers and the presence of mesenchymal cell markers on pulmonary pericytes. In the context of HDM-induced inflammation, pericytes demonstrated increased CXCR4 expression and migrated more readily to its cognate chemokine, CXCL12. This increase in migratory capacity was accompanied by pericyte accumulation in the airway wall, increased smooth muscle thickness, and symptoms of dyspnoea. Pericyte uncoupling from pulmonary vessels and subsequent migration to the airway wall were abrogated following intranasal delivery of the CXCL12 neutraligand LIT-927.
Conclusion: These results provide new insight into the role of the CXCL12/CXCR4 signaling axis in promoting pulmonary pericyte accumulation and airway remodeling and validate a novel target to address tissue remodeling associated with chronic inflammation.
CXCL12 Identifies Heterogeneity In Cortical Epithelial Cells
Andrea White, Sonia Parnell, Beth Lucas, Kieran James, Emilie Cosway, William Jenkinson, Graham Anderson. University of Birmingham.
Thymic epithelial cells can be sub divided into medullary and cortical epithelial cells, each playing a vital, but different role in the development of abT cells. More specifically CD4-CD8- T cells and CD4+CD8+ T cells can be found in the cortex and it’s the constant interactions with cortical epithelial cells through both b selection and positive selection that allows a normal program of development.
Whilst medullary epithelial cells (mTEC) are extremely well characterised, less is known about cortical epithelial cells (cTEC) and their heterogeneity. The CXCL12-CXCR4 axis has been shown to play a role in the positioning of CD4+CD8+ T cells within the cortex, and CXCL12 expression is widespread in cTEC.
We utilised the CXCL12dsRed reporter mouse, in which all cells express CXCL12 are dsRed+. We found in the neonate the majority of cTEC express CXCL12dsRed, but during ontogeny a progressive appearance of CXCL12dsRed- cTEC begins to emerge from 1 week postnatally and reaches a plateau at 10 weeks. Previous studies have shown CXCL12 is a Foxn1 dependant gene. Utilising both conventional PCR and bulk RNA sequencing we found that CXCL12dsRed- cTEC have reduced expression of Foxn1 and other Foxn1 dependant genes such PRSS16, PSMB11 and DDL4 when compared to CXCL12dsRed+ cTEC.
We show that positive selection using TCRa x CXCL12dsRed mice does not play a role in the appearance of CXCL12dsRed- cTEC, but b selection does with reduced appearance of CXCL12dsRed- cTEC compared to WT in Rag2-/- x CXCL12dsRed mice.
Collectively we are beginning to build a picture of the heterogeneity that is found in cTEC, and have identified a subset of cTEC that lack the expression of Foxn1 and other key known functional genes in cTEC. We have also shown that the appearance of CXCL12dsRed- cTEC could be in apart due to DN3a thymocytes as seen by the reduction in Rag2-/- x CXCL12dsRed mice.
Protein Kinase C-δ C2 Domain Interactions in Breast Cancer
Rasha Kahder and Lodewijk Dekker University of Nottingham
Protein kinase Cδ (PKCδ) has been previously targeted for breast cancer. It is extremely over expressed in breast cancer cells, especially the aggressive forms, over normal cells. The δC2 and δV5 domains have been reported to have essential roles in PKCδ activation and PKCδ-induced apoptosis. The two domains interact to stabilize the inactive conformation of PKCδ and retain its cytosolic localization. Upon stimulation, the two dissociate releasing the δV5 to bind importin-α and translocate to the nucleus, which initiates apoptosis. To maintain apoptosis, the domains reassociation needs to be blocked. A novel full-sequence inactive PKCδ model identified three δC2 target pockets with a potential to block the reassociation. Using High throughput screening, over one million compounds were docked in the target pockets in the active and inactive states of δC2, resulting in five HITs. Three breast cancer cell lines (MDA-MB-468, MDA-MB-231, and MCF-7) were transfected using pIRES‐myc‐δC2 plasmid to overexpress δC2. Compounds were assayed in non-transfected and transfected cell lines with and without H2O2 as a stimulus. The apoptotic effect was then assessed using flow cytometry in an annexin V-FITC apoptosis assay. Viability assays of the compounds, with and without stimuli, have shown that the compounds are promising inducers of PKCδ-mediated apoptosis in MDA-MB-468 and MCF-7. Cells stimulation with H2O2 enhances the compound-induced apoptosis. Four of the five compounds have shown superior apoptosis to comparable concentrations of Etoposide, Paclitaxel, and Cisplatin in MCF-7 and MDA-MB-468. A PKC-δ subcellular localization western blot is being carried to verify the mechanism of apoptosis.
Keynote Address
Professor Andrew MacDonald (The University of Manchester)
Dendritic cells and macrophages in promotion and regulation of pulmonary type 2 inflammation.
Dendritic cells and macrophages in promotion and regulation of pulmonary type-2 inflammation Fine control of immune cell activation is critical for preventing inflammatory disease, particularly in barrier sites such as the lung. However, the central mechanisms involved in controlling pulmonary macrophages and dendritic cells (DCs) during type-2 inflammation are currently poorly understood. We have assessed how lung macrophages respond during type-2 inflammation in vivo using administration of IL-4 complex (IL-4c), or following allergen exposure or helminth infection. In these settings, we have found that the lung environment dramatically impairs the ability of alveolar macrophages to respond and alternatively activate during type-2 inflammation, in comparison to macrophages from other tissue sites. We have interrogated the relative importance of glycolysis, mucins, surfactant and the host microbiota in regulating this process. In terms of DCs, using a mouse model of fungal allergic airway inflammation, we have found that conventional DCs are critical for promotion of antifungal allergic airway inflammation via activation of CD4+ T cells. Single cell RNA sequencing of DCs isolated from the lungs of mice exposed to fungal spores has revealed which DC subpopulations are associated with the induction of type-2 outcomes in this setting. Together, our data provide novel insight into the fundamental mechanisms that control macrophage and DC activation and function during pulmonary type-2 inflammation.
Flow Cytometry Applied To Cell Analysis And Therapies –Why Is There Variation In The Metrology?
Jon Petzing, Centre for Biological Engineering, Wolfson School of Mechanical, Electrical & Manufacturing Engineering, Loughborough University. j.petzing@lboro.ac.uk
Cell analysis and production of cell therapies, whether by single clinical or multiple biomanufacturing sites, introduces challenges to be solved to achieve the delivery of safe, viable diagnosis and therapeutics. From a biomanufacturing viewpoint, these challenges include the translation of the process from T-flask to bioreactor, inline measurement and assessment of the cell journey, concerns around infections and asepsis, correct cell differentiation, supply logistics, business considerations, and the quality and variation of starting materials. This latter component was investigated and considered in the context of Haematopoietic Stem Cells Therapy (HSCT) with orders of magnitude variation found within the literature. A single site clinical centre HSCT exemplar provided opportunity to further investigate the associated HSCT cell manipulation processes. It was observed that the Flow Cytometry processes using operator defined manual gating techniques was potentially prone to variation in data analysis as a function of changing operators. Bespoke studies were subsequently commissioned with 30+ operators being asked to manually gate and process increasingly complex cell data. The results demonstrated that as complexity increased then cell count data variation increased, but, if systematic protocols were put into place then this human operator variation could be mitigated.
Whilst manual gating is (in many cases) the preferred method for flow cytometry data processing, it was clear that a proliferation of unsupervised automated software platforms were being made increasingly available and competing for the data processing task. The aim of many of these software solutions is to reduce operator variation and increase the repeatability and accuracy of cell enumeration. Yet these software solutions involve a range of different mathematical paradigms that in themselves offer the potential for introducing different final cell count values and hence variation into the clinical and biomanufacturing setting. The research completed here has been to develop agnostic synthetic two and three cluster data sets that allows bespoke testing of a range of software platforms. This offers opportunity for unambiguous statements of accuracy and repeatability for each software solution, thus leading to a comparability analysis.
This presentation considers how variation in the metrology of flow cytometry (specifically the data analysis component) can potentially impact quality of diagnosis within the clinical setting, and also affect the decision making associated with cell therapy critical quality attributes from a biomanufacturing perspective.
Development of an In Vitro, Human, Cell-based Assay to Investigate the Role of Lipids and Invariant NKT cells in Allergic Sensitisation
Georgina V Hopkins1, Stella Cochrane2, David Onion1, and Lucy C Fairclough, 1
1School of Life Sciences, The University of Nottingham, Nottingham NG7 2UH, UK
2SEAC, Unilever, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ, UK
Abstract
Background
Immunoglobulin E (IgE)-mediated allergies are increasing in prevalence. However, the mechanisms underpinning the first phase of IgE-mediated allergy, allergic sensitisation, are still not clear. Recently, the potential involvement of lipids in allergic sensitisation has been proposed, with reports that they can bind allergenic proteins and act on immune cells to skew to a T helper type 2 (Th2) response. However, the existing research in this area is limited and predominantly uses murine models.
Objectives
The aim of this research it to develop an in vitro method to study the role of lipids and invariant natural killer T (iNKT) cells utilising a human model of allergic sensitisation.
Methods
Due to low abundance of iNKT cells in human peripheral blood, iNKT cells were expanded over 14 days by stimulation with the glycolipid, α-GalCer, which is the most potent activator of iNKT cells. The iNKT cells were then isolated and co-cultured with autologous, α-GalCer-pulsed dendritic cells (DCs) to allow iNKT-lipid binding. Th1 and Th2 iNKT cell cytokine release were then measured by flow cytometry. The internalisation of α-GalCer by DCs was confirmed by imaging cytometry.
Results
The results indicated α-GalCer induced an 80-fold expansion of iNKT cells by Day 14. Expansion with α-GalCer also induced a shift in iNKT cell phenotype from predominantly CD4-CD8- at Day 0, to predominantly CD4+CD8- iNKT cells by Day 14. The iNKTs co-cultured with autologous α-GalCer-pulsed DCs displayed increased IL-4 and IFN-y secretion within 5 hours of co-culture. Fluorescent α-GalCer was successfully internalised by immature DCs.
Conclusion
Overall, this establishes a human model system where allergen-associated lipids, such as peanut lipids, can be used in substitute of α-GalCer, to determine whether lipids enhance iNKT cell Th2 cytokine secretion, shifting towards a state of allergic sensitisation.
Physical activity is associated with reduced tethering and migration of pro-inflammatory monocytes in White European and South Asian males with and without central obesity.
Matthew J Roberts1,2, Malik Hamrouni1,2, Alex J Wadley3, Nicolette C Bishop1,2.
1 National Centre for Sport and Exercise Medicine, School of Sport, Exercise and Health Sciences, Loughborough University, Epinal Way, Loughborough, United Kingdom, LE11 3TU.
2 National Institute for Health Research (NIHR) Leicester Biomedical Research Centre, University Hospitals of Leicester, National Health Service (NHS) Trust and the University of Leicester, Leicester, United Kingdom, LE1 5WW.
3 School of Sport, Exercise & Rehabilitation Sciences, College of Life & Environmental Sciences, University of Birmingham, B15 2TT.
Migration of monocytes from the circulation into adipose tissue is a key event in developing chronic systemic inflammation in obesity and is related to increased cardiometabolic disease risk. Using an ex vivo model to mimic blood flow dynamics and endothelial integrin expression - coupled with flow cytometry - we previously reported monocyte adherence and migration is higher in men with central obesity versus lean men. Importantly, higher physical activity mitigated the effect of obesity on cell movement, independent of weight loss. We now wanted to investigate if this effect was influenced by ethnicity, as South Asians (SAs) have a higher risk for cardiometabolic disease than White Europeans (WEs).
Using the same ex vivo model (Figure 1) in SA and WE men with and without central obesity (N=10 in each group), we collected 1.75 x 105 PBMCs before migration (baseline) and tethered and migrated PBMCs migrating towards an adipose-tissue conditioned media. CD14 and CD16 expression (monocyte subsets) and chemokine receptor expression (CCR2, CCR5) was determined using four-colour flow cytometry.
Our preliminary evidence suggests monocyte tethering and migration towards adipose tissue are exacerbated in SA men with central obesity versus SA men who are lean and versus WE men with and without central obesity, independent of circulating monocyte numbers. Significantly, our data suggest that higher physical activity can mitigate the effect of obesity and ethnicity on cell movement. This is encouraging given the increased cardiometabolic disease risk, low levels of physical activity, and high levels of central adiposity in SAs.
Session 2
Tracking Host-Fungal Interactions In Vivo
Rebecca Drummond. Institute of Immunology & Immunotherapy / Institute of Microbiology & Infection, University of Birmingham.
Fungal infections of the central nervous system (CNS) are often life-threatening and typically affect patients with AIDS, CARD9 deficiency or iatrogenic immunosuppression. The most common cause of human fungal brain infection is Cryptococcus neoformans, which causes over 150,000 deaths each year. Our laboratory tries to understand how resident phagocytes within the brain, called microglia, recognise and respond to invading Cryptococcus fungi. We use fungal strains that express fluorescent proteins to track their interactions with host microglia, and use multi-colour flow cytometry to analyse the quantity and quality of such interactions in the brain. Using these approaches, we have found that microglia become hosts for intracellular fungal growth and that this interaction promotes brain fungal infection. We hypothesised that the fungus utilises microglia to gain access to restricted nutrients in the brain, such as copper. To test that, we generated a fluorescent-reporter fungal strain that switches on GFP expression when importing copper. Using flow cytometry, we found that GFP expression is higher in fungal cells that are bound within microglia, compared to ‘free’ fungal cells that are not associated with host cells. Therefore, Cryptococcus fungi upregulate copper importing machinery primarily within microglia, which explains the fungal dependence on microglia for early growth in the brain.
The effect of cigarette smoke on the production of extracellular vesicles by immune cells
Nancy Gomez1, David Onion1, Victoria James2 and Lucy Fairclough1
1School of Life Sciences, The University of Nottingham. 2School of Veterinary Medicine, The University of Nottingham
Smoking or other inhaled irritants induce an inflammatory response which plays a critical role in the initiation and progression of Chronic Obstructive Pulmonary Disease (COPD). Extracellular vesicles (EVs) originating from the airways have the potential to initiate and propagate inflammation in lung and systemic inflammatory conditions. The aim of this study is to examine the effect of cigarette smoke on the production of extracellular vesicles by cells of the immune system. Isolated peripheral blood mononuclear cells (PBMCs) were exposed to cigarette smoke extract (CSE) for 24 hours. After defining the presence of EVs by transmission electron microscopy (TEM), Calcein-AM and fluorophore-associated anti-tetraspanin monoclonal antibodies (moAbs) for CD9, CD63 and CD81 were added, and EVs/mL were measured by imaging flow cytometry (IFC). PBMC-derived EVs were isolated by size exclusion chromatography and were profiled using the MACSPlex exosome kit. The presence of EVs was confirmed by TEM. EV numbers and tetraspanins expression were identified by IFC, showing that EVs/mL increased with exposure to CSE and that addition of multiple anti-tetraspanin moAbs does not affect detection of any tetraspanin. Key cell markers on EV membranes for each CSE concentration were identified. This study demonstrates increased EVs release from PBMCs following exposure to CSE and shows the multiple tetraspanins and membrane markers on EVs. Future work will observe the effect of EV uptake on T cell phenotype.
Developing a flow cytometry based toolkit for assessing mesenchymal stromal cell functional properties
Jaspreet Bansal – Aston University
Mesenchymal stromal cells (MSC) are non-hematopoietic tissue resident progenitor cells and are a key mediator for cell based therapies. MSCs have low immunogenicity and crucially can both promote resolution of inflammatory conditions through modulation of immune cells as well as promoting tissue repair. However, MSCs are a rare population of cells in the body and require to be expanded ex vivo to provide the large number of cells required for therapy. Unfortunately, during expansion in culture, MSCs spontaneously differentiate, losing their clinically important repair and immuno-modulatory properties, instead become lineage committed stromal cells. Currently we cannot define clinically efficacious naïve multipotent, immuno-modulatory MSCs from their more mature progeny using phenotypic surface markers. Therefore, different forms of functional analysis have to be employed to determine the potential of laboratory expanded MSCs.
We have developed a flow cytometry based toolkit of functional measurements to assess changes in MSC physiology. This allows monitoring of distinct physiological processes known to correlate to MSC immuno-modulatory potential. Our workflow uses a toolkit to determine changes in metabolism (mitochondrial activity), senescence (b-Galactosidase activity, cell surface phenotype as well as the measuring the MSCs immuno-modulatory potential with immune cells (T cell suppression, induction of Tregs). We propose this flow cytometry based approach allows evaluation of multiple processes known to impact on MSC activity and is a useful strategy for measuring the effects of different culture conditions on MSC function, and how this may impact on their subsequent use for patients.
Session 3
CXCL12 drives pericyte accumulation and airway remodeling in allergic airway disease
Jill Johnson, College of Health and Life Sciences, Aston University.
Background: Airway remodeling is a significant contributor to impaired lung function in chronic allergic airway disease, and pulmonary pericytes have been shown to significantly contribute to airway wall remodeling and lung dysfunction in a mouse model of asthma. This study aimed to characterize pulmonary pericytes in healthy and inflamed lungs, and to elucidate the mechanism by which pericytes accumulate in the airway wall in a mouse model of chronic allergic airway inflammation driven by respiratory exposure to house dust mite (HDM) extract.
Methods: Magnetic sorting for CD146+ pericytes was performed on healthy mice, and these cells were then stained for cell surface markers using the Biolegend LegendScreenTM and assessed by flow cytometry. Subsequently, mice were subjected to a protocol of chronic airway inflammation driven by intranasal delivery of the common environmental aeroallergen HDM. Phenotypic changes to lung pericytes were assessed by flow cytometry and immunostaining, and the functional capacity of these cells was evaluated using in vitro migration assays. The molecular mechanisms driving these processes were targeted pharmacologically in vivo and in vitro using the CXCL12 neutraligand LIT-927.
Results The LegendScreenTM indicated the absence of hematopoietic cell markers and the presence of mesenchymal cell markers on pulmonary pericytes. In the context of HDM-induced inflammation, pericytes demonstrated increased CXCR4 expression and migrated more readily to its cognate chemokine, CXCL12. This increase in migratory capacity was accompanied by pericyte accumulation in the airway wall, increased smooth muscle thickness, and symptoms of dyspnoea. Pericyte uncoupling from pulmonary vessels and subsequent migration to the airway wall were abrogated following intranasal delivery of the CXCL12 neutraligand LIT-927.
Conclusion: These results provide new insight into the role of the CXCL12/CXCR4 signaling axis in promoting pulmonary pericyte accumulation and airway remodeling and validate a novel target to address tissue remodeling associated with chronic inflammation.
CXCL12 Identifies Heterogeneity In Cortical Epithelial Cells
Andrea White, Sonia Parnell, Beth Lucas, Kieran James, Emilie Cosway, William Jenkinson, Graham Anderson. University of Birmingham.
Thymic epithelial cells can be sub divided into medullary and cortical epithelial cells, each playing a vital, but different role in the development of abT cells. More specifically CD4-CD8- T cells and CD4+CD8+ T cells can be found in the cortex and it’s the constant interactions with cortical epithelial cells through both b selection and positive selection that allows a normal program of development.
Whilst medullary epithelial cells (mTEC) are extremely well characterised, less is known about cortical epithelial cells (cTEC) and their heterogeneity. The CXCL12-CXCR4 axis has been shown to play a role in the positioning of CD4+CD8+ T cells within the cortex, and CXCL12 expression is widespread in cTEC.
We utilised the CXCL12dsRed reporter mouse, in which all cells express CXCL12 are dsRed+. We found in the neonate the majority of cTEC express CXCL12dsRed, but during ontogeny a progressive appearance of CXCL12dsRed- cTEC begins to emerge from 1 week postnatally and reaches a plateau at 10 weeks. Previous studies have shown CXCL12 is a Foxn1 dependant gene. Utilising both conventional PCR and bulk RNA sequencing we found that CXCL12dsRed- cTEC have reduced expression of Foxn1 and other Foxn1 dependant genes such PRSS16, PSMB11 and DDL4 when compared to CXCL12dsRed+ cTEC.
We show that positive selection using TCRa x CXCL12dsRed mice does not play a role in the appearance of CXCL12dsRed- cTEC, but b selection does with reduced appearance of CXCL12dsRed- cTEC compared to WT in Rag2-/- x CXCL12dsRed mice.
Collectively we are beginning to build a picture of the heterogeneity that is found in cTEC, and have identified a subset of cTEC that lack the expression of Foxn1 and other key known functional genes in cTEC. We have also shown that the appearance of CXCL12dsRed- cTEC could be in apart due to DN3a thymocytes as seen by the reduction in Rag2-/- x CXCL12dsRed mice.
Protein Kinase C-δ C2 Domain Interactions in Breast Cancer
Rasha Kahder and Lodewijk Dekker University of Nottingham
Protein kinase Cδ (PKCδ) has been previously targeted for breast cancer. It is extremely over expressed in breast cancer cells, especially the aggressive forms, over normal cells. The δC2 and δV5 domains have been reported to have essential roles in PKCδ activation and PKCδ-induced apoptosis. The two domains interact to stabilize the inactive conformation of PKCδ and retain its cytosolic localization. Upon stimulation, the two dissociate releasing the δV5 to bind importin-α and translocate to the nucleus, which initiates apoptosis. To maintain apoptosis, the domains reassociation needs to be blocked. A novel full-sequence inactive PKCδ model identified three δC2 target pockets with a potential to block the reassociation. Using High throughput screening, over one million compounds were docked in the target pockets in the active and inactive states of δC2, resulting in five HITs. Three breast cancer cell lines (MDA-MB-468, MDA-MB-231, and MCF-7) were transfected using pIRES‐myc‐δC2 plasmid to overexpress δC2. Compounds were assayed in non-transfected and transfected cell lines with and without H2O2 as a stimulus. The apoptotic effect was then assessed using flow cytometry in an annexin V-FITC apoptosis assay. Viability assays of the compounds, with and without stimuli, have shown that the compounds are promising inducers of PKCδ-mediated apoptosis in MDA-MB-468 and MCF-7. Cells stimulation with H2O2 enhances the compound-induced apoptosis. Four of the five compounds have shown superior apoptosis to comparable concentrations of Etoposide, Paclitaxel, and Cisplatin in MCF-7 and MDA-MB-468. A PKC-δ subcellular localization western blot is being carried to verify the mechanism of apoptosis.
Keynote Address
Professor Andrew MacDonald (The University of Manchester)
Dendritic cells and macrophages in promotion and regulation of pulmonary type 2 inflammation.
Dendritic cells and macrophages in promotion and regulation of pulmonary type-2 inflammation Fine control of immune cell activation is critical for preventing inflammatory disease, particularly in barrier sites such as the lung. However, the central mechanisms involved in controlling pulmonary macrophages and dendritic cells (DCs) during type-2 inflammation are currently poorly understood. We have assessed how lung macrophages respond during type-2 inflammation in vivo using administration of IL-4 complex (IL-4c), or following allergen exposure or helminth infection. In these settings, we have found that the lung environment dramatically impairs the ability of alveolar macrophages to respond and alternatively activate during type-2 inflammation, in comparison to macrophages from other tissue sites. We have interrogated the relative importance of glycolysis, mucins, surfactant and the host microbiota in regulating this process. In terms of DCs, using a mouse model of fungal allergic airway inflammation, we have found that conventional DCs are critical for promotion of antifungal allergic airway inflammation via activation of CD4+ T cells. Single cell RNA sequencing of DCs isolated from the lungs of mice exposed to fungal spores has revealed which DC subpopulations are associated with the induction of type-2 outcomes in this setting. Together, our data provide novel insight into the fundamental mechanisms that control macrophage and DC activation and function during pulmonary type-2 inflammation.
Poster Abstracts
1. Applying Uncertainty Analysis To Assess The Variation Of Operator Performance When Manually Gating Flow Cytometry Data Rebecca Grant1, Karen Coopman1, Sandro Gomes2, Jonathan Campbell3, Julian Braybrook3, Jon Petzing1*
1Centre for Biological Engineering, Loughborough University 2GlaxoSmithKline, Gunnels Wood Road, Stevenage, 3National Measurement Laboratory, LGC, Teddington *Corresponding author: j.petzing@lboro.ac.uk
The use of flow cytometry (FC) for cell identification and counting is prevalent within the biomanufacture of Advanced Therapy Medicinal Products (ATMPs) both in clinical centres and biomanufacturing facilities. Operators involved in the use of FC and the analysis of the resultant datasets have the option of manually gating cell clusters or using a range of automated software platforms (supplied by FC instrument manufacturers, or third party). It is well known that many operators prefer to manually gate on the basis of speed and simplicity, however with increasing data complexity (especially when dealing with multi-colour FC instruments) there is significant potential for variation in final cell count to increase as a function of operator subjectivity.
Understanding this relationship between increasing data dimensionality and operator subjectivity during manual gating techniques is important because it allows process facilities to comprehend and quantify the potential for process error as a function of incorrect measurement data. This research has specifically considered how uncertainty analysis can be used to better define operator variation over and beyond traditional measures such as the Coefficient of Variation (CV), where CV is typically only representative of the final gating process and does not provide resolution of understanding at each gating step [1, 2].
Three Stages of cell model complexity were designed and trialled, measuring variation in manual gating sequence steps to obtain a target cell population, with operators selected from academic and industrial research, and national measurement laboratory process team environments. Increasing operator variation was found as a function of increasing FC data complexity. When considering 3 step, 5 step and 8 step FC analysis, operator CV was found to range from 6% to 57% (3 step to 8 step data respectively) [3].
At the same time uncertainty analysis was applied to each gating analysis step providing clear definition of variation at each step allowing the generation of inter- and intra-operator variation statistics. Resultant combined uncertainty statements expressed at a one sigma coverage factor (67%) showed variation from 12% (3 step data) through to 34% (8 step data). Process improvement for manually gated FC analysis can therefore take advantage of this more detailed variation analysis framework and provides a benchmark for comparison with FC automated software platforms. In addition, this investigation will be pertinent when defining the human factors requirement for manual gating and evaluation of cell metrics, both for biomanufacturing and clinical scenarios, as well as helping to inform regulatory science.
2. Defining the surface proteome of extracellular vesicles via nanoflow cytometry
Rachel Butler1, Alan Goddard2, Dimitri Aubert3 and Andrew Devitt1
1School of Biosciences, Health and Life Sciences, Aston University, UK
2Aston Centre for Membrane Proteins and Lipids Research (AMPL), Aston University, UK
3NanoFCM Co. Ltd, D6 Thane Road, Nottingham, UK
As cells die in vivo they are removed by phagocytes in a process that resolves inflammation and prevents disease. For clearance of dying (apoptotic) cells to be efficient and timely, dying cells release extracellular vesicles (EV) to attract phagocytes to sites of cell death. Crucially, this interaction of EV with the immune system underpins the control of inflammation, a process central to health, regenerative medicine and many inflammatory diseases.
Multiparametric single-EV analysis is a next generation approach enabling in-depth characterisation and identification of novel EV subpopulations. A significant challenge, however, is EV heterogeneity. In particular, EV vary greatly in size (<50nm - >1µm). NanoFCM have developed a novel platform that combines the capabilities of flow cytometry at the nano-scale with high sensitivity (40nm limit of scatter detection) and specificity (simultaneous scatter and fluorescence detection). This will enable comprehensive EV phenotyping at a scale that has previously been difficult to detect. Through collaboration between Aston University and NanoFCM, the EV surface will be probed at high resolution to establish a fundamental atlas of the surface proteome of apoptotic cell-derived EVs (ACdEVs) across the entire size range.
ACdEV surface proteome will be linked to function as we address the following key questions: what are the key components of ACdEV that enable communication with the immune system? Do ACdEV of differing sizes and sources interact in a similar manner with the immune system? Is it possible to produce a synthetic ACdEV with a defined surface proteome to act as a mimic of ACdEV function?
3. Protocatechuic acid and vanillic acid, phenolic metabolites of cyanidin-3-glucoside, prevent initial stages of atherosclerosis Joseph Festa1, Harprit Singh1, Aamir Hussain1,2, Amon B Hackney1, Tarsem Sahota3 & Mariasole Da Boit1
1.Leicester School of Allied Health Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK
2.Department of Respiratory Sciences, College of Life Sciences, University of Leicester, Leicester, LE1 7RH, United Kingdom
3.Leicester school of Pharmacy, faculty of health and life sciences De Montfort University, the gateway Leicester
Introduction: Cyandin-3-Glucoside (C3G) is a major anthocyanin found in a variety of foods and it is known to have health promoting effect specifically within the vasculature. However, anthocyanins are usually transformed by the human gut to metabolite products reaching plasmatic concentration much higher than their original form, potentially indicating their contribution to the biological activity previously observed with C3G.
Purpose: The main aim of this study was to assess C3G, protocatechuic acid (PCA) and vanillic acid (VA) effect on an in vitro model replicating the initial stages of atherosclerosis.
Methods: Primary Human Umbilical Vein Endothelial Cells (HUVEC) were cultured +/- monocytes (THP-1) cells and were pre-treated with either C3G, PCA and VA 5 μM for 24-hr then stimulated with or without TNF-α 10 ng/ml for an additional 24-hr. After treatments cells were washed in PBS, trypsinized with 0.25% Trypsin-EDTA (Sigma), and stained with either Annexin V and propidium iodide, VCAM-1 (CD106) or DCFHDA. Cells were then fixed with 2% FBS acquired for plate reader or flow cytometry.
Results: All treatments prevented TNF-α induced monocyte adhesion (P<0.05). This effect may have been a result of treatments decreasing the % of apoptosis, CD106 production in HUVECs (P<0.05) although only PCA and VA reduced ROS production.
Discussion: In the present study, we demonstrated that pre-exposure of endothelial cells to phenolic metabolites prevented the induction of monocytes adherence to endothelial cells. This was associated with a reduction of apoptosis, production of ROS and a % decrease in CD106 which could explain the findings of this study.
4. Full Spectrum Flow Cytometry with the Sony ID7000
Maria Isabel Haig, University of Nottingham Flow Cytometry Facility
Spectral flow cytometry is an emerging technology that, by enabling high-dimensional fluorescent flow cytometry, offers a revolution in resolution and the number of parameters that can be measured simultaneously. Conventional flow cytometry uses mirrors and band pass filters to select specific bands of the optical spectrum for detection, whereas full spectrum flow cytometers, capture a unique emission spectrum for each fluorophore. The improved capabilities and precision of this analytical platform, compared with previous generations of conventional flow cytometers, enable the ability to resolve highly overlapping dyes, that could not previously be used together. This will enable the ‘undoable’ combinations such as GFP/YFP and AF647/APC and by extension allow researchers to build highly multiparametric panels.
5. Novel 355nm (and Lower) Excitable and Tuneable Emission (Blue through Red) Fluorophores Utilised in Flow Cytometry
Sareena Sund,a Sunil Claire,b Parvez Iqbal,b Michael Butlin, a Owen Jones,a Karolis Virzbickas a,b and Jon A. Preece a,b,*
aSchool of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT
bChromaTwist, Birmingham Research Park, Vincent Drive, Edgbaston, Birmingham B15 2SQ
Labelling antibodies with a fluorescent reporter molecule allows for the detection and localisation of antigens situated within a cell, tissue or organ. An array of fluorescent probes can be attached to different antibodies, each conveying its own distinct spectral properties that can be exploited by flow cytometry and multiphoton microscopy. We have, to date, >50 novel dyes that have an excitation profile from ~250nm to ~420 nm. The dyes can be chemically tuned to emit from 400 nm through to 630 nm,1, 2 and hence have utility to increase multiplexing in both multicolour and spectral flow cytometry.
This study focuses on antibody conjugation of one dye via the formation of an active ester on a novel dye (Gen-2-Dye). The reaction uses EDC/sulfo-NHS chemistry to activate the carboxylic acid functionality on the fluorophore which enables conjugation to a CD8 antibody via an amide linkage. Characterisation by UV-Vis absorbance and steady-state emission spectroscopy confirmed the presence of the antibody with the distinct fluorescent signature of the Gen-2-Dye. A commercial antibody conjugation check kit also confirmed successful conjugation and antibody functionality (Figure 1a).
The Gen-2-Dye•CD8 conjugate was used to stain compensation beads and displayed a bimodal distribution in the bead population relative to the unstained control (Figure 1b-d). Further optimisation is underway to achieve two distinctly separated populations of
negative and positively stained compensation beads.
Workshops
Workshop 1
Beginners 1
Lindsay Bentley (Miltenyi Biotech) - Improving flow analysis through sample prep.
Generating good quality data from flow analysis requires the input of good quality samples. Miltenyi Biotec offers a range of products designed for the optimisation of sample preparation whether from blood or tissue samples, all with the aim of getting the best data possible from flow cytometric analysis. I’ll also discuss our REAlease antibody technology which allows for the removal of labels from cells after sorting allowing for flexibility in downstream processing.
Stephen Rackstraw (ThermoFisher) – Why are researchers excited about spectral flow cytometry?
Learn how spectral flow cytometry enables researchers to get more information about their cells in a single flow cytometry experiment).
Advanced 1
Vendula Sinkorova (SONY) – Spectral Cell Analysis and its advantages.
As a technology, Flow Cytometry provides rapid, multi-parametric analysis of fluorescence-labelled single particles in suspension. Flow cytometers utilize lasers as light source, producing both scattered and fluorescent light signals that are read by detectors, such as photodiodes or photomultiplier tubes. Spectral flow cytometers diffract fluorescence emitted by the measured particles and direct it into many fluorescent detectors placed all over the spectrum. Because of the full spectrum analysis, more signal is processed, and more information is gained. This way of signal processing allows us to avoid signal loss due to light filtering and compensation and gives us more freedom in experiment design.
During this workshop, the Senior Application Scientist of Sony Biotechnology Vendula Sinkorova, will cover the differences between Spectral and Conventional Flow Cytometry. She will give an overview of how spectral data is generated and how spectral unmixing works. You will learn how to read spectral plots and how to unmix autofluorescence, which no longer has to be just a background noise but an additional parameter, providing you with more in-depth understanding of your samples. We will talk about how looking at the full spectrum can provide us with more flexibility in panel design and highlight the potential advantages to your research.
Alice Law (NanoFCM) – NanoAnalyzer: Exploring the nano-sized with a dedicated flow cytometry platform.
Conventional flow cytometers often struggle to meet the sensitivity requirements for the analysis of nanoscale particles, such as exosomes, nanomedicine, and viruses. To meet this challenge, NanoFCM has developed the NanoAnalyzer, a dedicated nano-flow cytometry platform, which offers a flexible and high-throughput solution for sub-micron analysis.
Workshop 2
Beginners 2
Mike Blundell (Biorad) - How to avoid common pitfalls to get publication quality flow cytometry data.
This talk aims to walk you through some of the easy mistakes and common problems that you can face when trying to get meaningful and publication quality flow cytometry data including what measures you can take to avoid them. Topics include sample preparation, instrument set-up, sample acquisition, reagent choice and use, controls and data analysis. Find out how just a few simple steps can help avoid wasting precious samples and reagents and improve the quality of your data.
Adam Davison (Cytek) – Full Spectrum Profiling™ (FSP) – Best practices in reference control selection, optimisation and longitudinal performance validation.
Appropriate single stain control selection for the determination of spectral spillover is essential to ensure best possible outcomes in antigen assessment in multi-colour labelled samples. Here we will cover the core tenets of reference control selection and describe how these continue to be true in spectral cytometry as they are to classical flow cytometry. We will show how Full Spectrum Profiling™ (FSP) can rapidly allow for the determination of reference control quality and facilitate troubleshooting of reference control performance when compared to classical flow cytometry. We will introduce the Similarity™ Index and Complexity™ Index tools, embedded in the SpectroFlo® software, and show how these can be used to evaluate reference control performance and longitudinally track this over time. Through FSP we can introduce improvements to assay quality control of our reagents to ensure the robustness of data output in longitudinal flow cytometry studies.
Idhnan Hussain (CURIOX) - Next-Generation cell preparation with laminar wash technology.
Curiox Biosystems is committed to enabling accurate biological analysis through automation. Next-generation methods and applications--including single-cell 'omics technologies, flow cytometry, immunotherapy, and cell and gene therapy--deserve a next-generation approach to preparing samples for analysis. That’s why Curiox invented Laminar Wash technology, the only sample preparation method that eliminates centrifugation and the problems it introduces. The methodology keeps cells in their native state, improves cell retention and viability, and enhances reproducibility and miniaturisation, ultimately leading to reduced costs and workflow standardisation.
Advanced 2
Thomas Adejumo (Fluidigm) - How to set-up and run high parameter cytometry experiments.
An introduction into the operation and daily set-up of the new CyTOF XT high dimensional cytometer and how to reduce experimental viability and batch effects with proper high dimensional panel design and barcoding options.
Pj Chana - (Luminex) Join the revolution… Demonstrating how AI can simplify & enhance complex Imaging Flow Cytometry analysis.
Over the years Imaging flow cytometry has brought quantifiable measurements to microscopy data and expanded the repertoire of technologies under the flow cytometry banner. The high content nature of the data has allowed additional insight in many areas but, can also require an intimate understanding of the comprehensive, quantitative imaging software tools. This can prove to be a stumbling block.
Recent advances in artificial intelligence now allow researchers to leverage the power of cutting edge image recognition software which not only makes analysis far easier but also allows additional discovery from new and existing datasets.
Let us take you on a journey from visual qualification to statistical quantification using the new and improved Amnis AI suite.
Morgan Blaylock (BD Biosciences) - Road to Complex Biology: streamlining the power of high-dimensional biology with BD CellView™ and BD Rhapsody™.
Get Ready for Image Analysis Fast Enough for Cell Sorting. Discover with confidence by leveraging real time visual inspection of flow events, comprehensive cell characterization and precise image-based sorting.
BD CellView™ Image Technology is a novel high-speed cell imaging technology that empowers scientists to answer previously out of reach biological questions by amplifying the power of cell sorting and analysis through real-time integration of image and flow data. Get precise cellular insights that drive breakthrough discoveries.
Abstracts will appear here
Beginners 1
Lindsay Bentley (Miltenyi Biotech) - Improving flow analysis through sample prep.
Generating good quality data from flow analysis requires the input of good quality samples. Miltenyi Biotec offers a range of products designed for the optimisation of sample preparation whether from blood or tissue samples, all with the aim of getting the best data possible from flow cytometric analysis. I’ll also discuss our REAlease antibody technology which allows for the removal of labels from cells after sorting allowing for flexibility in downstream processing.
Stephen Rackstraw (ThermoFisher) – Why are researchers excited about spectral flow cytometry?
Learn how spectral flow cytometry enables researchers to get more information about their cells in a single flow cytometry experiment).
Advanced 1
Vendula Sinkorova (SONY) – Spectral Cell Analysis and its advantages.
As a technology, Flow Cytometry provides rapid, multi-parametric analysis of fluorescence-labelled single particles in suspension. Flow cytometers utilize lasers as light source, producing both scattered and fluorescent light signals that are read by detectors, such as photodiodes or photomultiplier tubes. Spectral flow cytometers diffract fluorescence emitted by the measured particles and direct it into many fluorescent detectors placed all over the spectrum. Because of the full spectrum analysis, more signal is processed, and more information is gained. This way of signal processing allows us to avoid signal loss due to light filtering and compensation and gives us more freedom in experiment design.
During this workshop, the Senior Application Scientist of Sony Biotechnology Vendula Sinkorova, will cover the differences between Spectral and Conventional Flow Cytometry. She will give an overview of how spectral data is generated and how spectral unmixing works. You will learn how to read spectral plots and how to unmix autofluorescence, which no longer has to be just a background noise but an additional parameter, providing you with more in-depth understanding of your samples. We will talk about how looking at the full spectrum can provide us with more flexibility in panel design and highlight the potential advantages to your research.
Alice Law (NanoFCM) – NanoAnalyzer: Exploring the nano-sized with a dedicated flow cytometry platform.
Conventional flow cytometers often struggle to meet the sensitivity requirements for the analysis of nanoscale particles, such as exosomes, nanomedicine, and viruses. To meet this challenge, NanoFCM has developed the NanoAnalyzer, a dedicated nano-flow cytometry platform, which offers a flexible and high-throughput solution for sub-micron analysis.
Workshop 2
Beginners 2
Mike Blundell (Biorad) - How to avoid common pitfalls to get publication quality flow cytometry data.
This talk aims to walk you through some of the easy mistakes and common problems that you can face when trying to get meaningful and publication quality flow cytometry data including what measures you can take to avoid them. Topics include sample preparation, instrument set-up, sample acquisition, reagent choice and use, controls and data analysis. Find out how just a few simple steps can help avoid wasting precious samples and reagents and improve the quality of your data.
Adam Davison (Cytek) – Full Spectrum Profiling™ (FSP) – Best practices in reference control selection, optimisation and longitudinal performance validation.
Appropriate single stain control selection for the determination of spectral spillover is essential to ensure best possible outcomes in antigen assessment in multi-colour labelled samples. Here we will cover the core tenets of reference control selection and describe how these continue to be true in spectral cytometry as they are to classical flow cytometry. We will show how Full Spectrum Profiling™ (FSP) can rapidly allow for the determination of reference control quality and facilitate troubleshooting of reference control performance when compared to classical flow cytometry. We will introduce the Similarity™ Index and Complexity™ Index tools, embedded in the SpectroFlo® software, and show how these can be used to evaluate reference control performance and longitudinally track this over time. Through FSP we can introduce improvements to assay quality control of our reagents to ensure the robustness of data output in longitudinal flow cytometry studies.
Idhnan Hussain (CURIOX) - Next-Generation cell preparation with laminar wash technology.
Curiox Biosystems is committed to enabling accurate biological analysis through automation. Next-generation methods and applications--including single-cell 'omics technologies, flow cytometry, immunotherapy, and cell and gene therapy--deserve a next-generation approach to preparing samples for analysis. That’s why Curiox invented Laminar Wash technology, the only sample preparation method that eliminates centrifugation and the problems it introduces. The methodology keeps cells in their native state, improves cell retention and viability, and enhances reproducibility and miniaturisation, ultimately leading to reduced costs and workflow standardisation.
Advanced 2
Thomas Adejumo (Fluidigm) - How to set-up and run high parameter cytometry experiments.
An introduction into the operation and daily set-up of the new CyTOF XT high dimensional cytometer and how to reduce experimental viability and batch effects with proper high dimensional panel design and barcoding options.
Pj Chana - (Luminex) Join the revolution… Demonstrating how AI can simplify & enhance complex Imaging Flow Cytometry analysis.
Over the years Imaging flow cytometry has brought quantifiable measurements to microscopy data and expanded the repertoire of technologies under the flow cytometry banner. The high content nature of the data has allowed additional insight in many areas but, can also require an intimate understanding of the comprehensive, quantitative imaging software tools. This can prove to be a stumbling block.
Recent advances in artificial intelligence now allow researchers to leverage the power of cutting edge image recognition software which not only makes analysis far easier but also allows additional discovery from new and existing datasets.
Let us take you on a journey from visual qualification to statistical quantification using the new and improved Amnis AI suite.
Morgan Blaylock (BD Biosciences) - Road to Complex Biology: streamlining the power of high-dimensional biology with BD CellView™ and BD Rhapsody™.
Get Ready for Image Analysis Fast Enough for Cell Sorting. Discover with confidence by leveraging real time visual inspection of flow events, comprehensive cell characterization and precise image-based sorting.
BD CellView™ Image Technology is a novel high-speed cell imaging technology that empowers scientists to answer previously out of reach biological questions by amplifying the power of cell sorting and analysis through real-time integration of image and flow data. Get precise cellular insights that drive breakthrough discoveries.
Abstracts will appear here