AAI Immunology 2018

AAI Immunology 2018

4 May 2018

AAI 2018

Sartorius Booth #1136

Visit our booth at AAI to learn how the IncuCyte® S3 Live-Cell Analysis System and the Intellicyt iQue® Screener PLUS expand your in vitro assay toolkit for immunology research. These groundbreaking systems provide information-rich solutions that deliver new insights into biological mechanisms at unprecedented speed, depth and scale to accelerate immunologic discovery and reveal the dynamic and complex nature of immune cell biology.

From tracking biology over time to providing rapid assessment of immune cell phenotype, activation and function, to assessing the dynamics of cancer and immune cell interactions, these powerful technologies deliver the deep insight that complex immune biology demands.

Stop by for a chance to win prizes!

At the show, please join our exhibitor workshop, presentation and posters.

LEARN MORE ABOUT IMMUNOLOGY 2018


Workshop:
Characterizing functions of immune cells using live-cell analysis


Presenters:

  • Chia-Wei Li, Instructor, Dept. of Molecular and Cellular Oncology, MD Anderson Cancer Center
  • Gillian Lovell, Ph.D., Senior Scientist, Sartorius
  • Lindy O’Clair, Product Manager, Sartorius

Date and Time: Monday, May 7, 2018 1:45 PM – 2:30 PM

Room: 2

Join this workshop to learn how kinetic, image-based analysis of living immune cells innovates discoveries by providing deeper, more meaningful and biologically relevant information about immune cell biology, function and dynamics. The workshop will highlight research done by Dr. Chia-Wei Li from MD Anderson Cancer Center, revealing the molecular mechanism of PD-L1 regulation in cancer cells, utilizing real-time analysis to evaluate T cell-mediated cancer cell killing effect. Join us to learn more about real-time, live-cell assays for various cell types and co-culture models as well as new reagents and software tools for live-cell immunocytochemistry and immunophenotyping.


Presentation: Innate Leukocyte Responses

Presenter: Dr. Gillian Lovell, Senior Scientist
Date and Time: Tuesday, May 8, 2018 11:45 AM - 12:00 PM
Room: 17AB


Posters:

Real-time visualisation and quantification of Neutrophil Extracellular Traps

Presenter: Dr. Gillian Lovell, Senior Scientist
Date and Time: Saturday, May 5, 2018 2:30 PM - 3:45 PM
Room: Exhibit/Poster Hall
Program No: 49.5 (INC.546)
Authors: G. Lovell, N. Bevan, T. Dale, D. Trezise. Essen BioScience Ltd, Biopark, Welwyn Garden City, Hertfordshire UK AL7 3AX

The ability of neutrophils to release extracellular traps (NETs) is one of several mechanisms by which the body defends against infection.  When neutrophils encounter invading pathogens, the cells release a mixture of antimicrobial proteins and chromatin to trap and degrade microbes. NETs have been implicated in a number of disorders including atherosclerosis, systemic lupus erythematosus (SLE) and thrombosis.

NETosis can be stimulated in vitro using a number of methods including chemical compounds, microbes and microcrystals. In order to better understand the signaling pathways involved, we developed a fully kinetic live-cell imaging assay for NETosis (96-well format, IncuCyte S3).  Using a fluorescent cell impermeant DNA-binding reagent (IncuCyte CytoTox Green), NET release was visualised and quantified in real time. In both primary human neutrophils and differentiated ‘neutrophil-like’ dHL60 cells, PMA (100nM) induced rapid (2h onset, peak 4-6h), time-dependent increase in fluorescence and nuclear degradation. A concomitant increase in myeloperoxidase, elastase (immunofluorescence) and cell-free DNA (Picogreen) was observed, validating the NETosis signal. PMA-induced NETosis was ROS-dependent (CellRox Red) but did not cause externalisation of phosphatidylserine (PS, Annexin V). In contrast, ionomycin (5mM) induced NETosis more rapidly (onset time <15’), did not induce ROS but did externalise PS. Neutrophils and dHL60 cells were both able to phagocytose bacterial bioparticles (IncuCyte pHrodo-E-coli). Taken together, these data illustrate how NETs and neutrophil signaling pathways can be robustly, meaningfully and efficiently analysed using automated live-cell imaging and compatible detection reagents.


Use of fluorescent Fab/Ab complexes and IncuCyte live-cell analysis to dynamically track cell surface markers and cell populations in mixed cultures

Presenter: Lindy O’Clair, Product Manager
Date and Time: Saturday, May 5, 2018 2:30 PM - 3:45 PM
Room: Exhibit/Poster Hall
Program No: 46.11 (IRC.442)

Authors: N. Bevan, V. Blancheteau, H. Campwala, N. Dana, N. Holtz, E. Endsley, T. Dale, G. Lovell, B. O’Clair, D. Trezise. Essen BioScience, Welwyn Garden City, Hertfordshire UK & Ann Arbor, Michigan, USA

Fluorescently-labelled antibodies are widely used for visualising cellular protein expression /distribution (e.g. immunocytochemistry) and immuno-phenotyping (e.g. flow cytometry). However, their applications are largely confined to single time-point (‘end’) or short term (min-h) detection studies, and the cell processing and labelling steps that are required often perturb the biology of interest.

To address this, and to enable long-term, non-invasive identification and quantification of cells in mixed cultures, we have developed a novel live-cell analysis strategy based on fluorescently-labelled antibody fragments (Fabs). An Fc-targeted anti-mouse Fab fragment was conjugated to a CF-488A (green) fluoroprobe (Biotium) and used to fluorescently tag Abs to cell surface markers via a simple one-step, mix, no-wash protocol. Addition of the FabFluor-Ab complex to living cells in full media/serum produced fluorescent labelling that was sufficiently bright and stable to allow repeated measurements for up to 5 days (IncuCyte S3). The Fab-Fluor/Ab/blocking dye combination did not perturb cell morphology or growth, with exception of known cytotoxic Abs. As proof of concept, PBMC immune cells were labelled with FabFlour/Ab complexes to CD4, CD8, CD20, CD45 and CD71 and tracked over time. The anticipated fraction of cells were labelled with each Ab, and activation of the PBMCs with CD3/IL-2 caused marked time-dependent expression of CD71. In another example, upregulation of the cell-surface checkpoint protein PDL-1 was measured over 48h in IFN-gamma-treated SKOV-3 and MDA-MB231 tumour cells. This FabFluor approach enables long term tracking and quantification of cell surface protein expression and the ability to identify cell subsets (cf flow cytometry) in living cultures and over time. This method should prove powerful in analyses on complex and advanced heterogeneous cell models.

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