Assays for Immuno-oncology Research Real-time automated measurements of immune and tumor cell dynamics within your incubator

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1 INCUCYTE LIVE-CELL ANALYSIS SYSTEM Assays for Immuno-oncology Research Real-time automated measurements of immune and tumor cell dynamics within your incubator

2 See what your cells are doing and when they do it Real-time tumor and immune cell analysis inside your incubator Gain insight into the dynamic biological processes that drive the immune response against cancer with the IncuCyte Live-cell Analysis System. Continuously monitor and analyze key events, from proliferation and activation, to migration and immune cell killing - without disturbing or exhausting your cells, or defining assay end-points. Reveal the interplay between immune and tumor cells Generate non-invasive imagebased measurements and movies of dynamic immune/ tumor cell-interactions Assess morphology and visually confirm interactions in situ in your incubator Get the answers you need Never miss a data point with real-time continuous analysis Analyze cells as they stay stationary in a physiologically-relevant, stable environment Save time and money Reduce time spent troubleshooting experiments with IncuCyte reagents, consumables and protocols Preserve your precious cells with assays that use low cell numbers Enjoy walk-away convenience as images are automatically acquired and analyzed Protect your cells Maintain cell health and morphology with non-perturbing reagent formulations Reduce manipulations and loss of cells with mix-andread reagents Study key steps with IncuCyte applications, reagents and consumables Effective immunotherapies require the immune system to recognize, engage, kill, and remove unwanted tumor cells. IncuCyte provides a range of applications,reagents, and consumables to study the key steps. Direct functional readouts from your cultures No cell lifting Visualize dynamic interactions between immune and tumor cells Study immune cell mobilization against cancer Characterize therapeutic effects on tumor cell killing and clearance Immune cell clustering & proliferation Monitor immune cell activation in real time Chemotaxis Measure immune cell migration towards tumor associated chemokines Immune cell killing Visualize and automatically analyze immune cell killing of tumor cells Phagocytosis Real-time analysis of tumor cell internalization using ph sensitive probes Customer testimonial Invaluable... easily monitor the effect of compounds or shrnas on cell growth over time very useful for looking at the effect of CD8 + T cell killing on both proliferation and apoptosis in the same well. Amanda Lennon, Sanofi 2

3 Monitor and analyze immune cell activation and inhibition in real-time Continuous live-cell proliferation, clustering and viability assays for T cells, PBMCs, monocytes and B cells. Automatically monitor immune cell activation without labelling, washing or cell lifting. Easily harvest cells or supernatants for complimentary downstream flow cytometry or chemokine analyses. L-kynurenine (L-kyn) inhibits T cell activation enabling cancer cells to escape immune surveillance. Label-free confluence measurements and cluster counts show a clear time-dependent, concentration-related inhibition of IL-2/ anti-cd3 activated T cell proliferation and clustering with the addition of exogenous L-kyn, a metabolite that blocks antigen-driven T cell proliferation and induces T cell death. Observe and measure immune cell migration towards tumor-associated chemokines Visually confirm and automatically measure migration towards chemoattractants with the IncuCyte Chemotaxis Cell Migration Software Module and ClearView 96-well Chemotaxis Plates Measure pro-/anti-migration effects and transendolthelial migration across HUVEC monolayers IncuCyte ClearView 96-well chemotaxis plates (above). Enable visualization and label free analysis of cell movement across the ClearView membrane insert (e.g. red arrow) through laseretched pores (yellow circles) towards chemoattractant in the reservoir plate. CXCR4-receptor mediated chemotaxis of human T cells (right). ClearView chemotaxis assay reveals CD3/CD28 activated T cell migration towards the CXCR4-receptor ligand SDF-1α as both chemotaxis and extravasation through a HUVEC monolayer. 3

4 Visualize and quantify immune cell killing Highly flexible assays with protocols developed for PBMCs and cytotoxic T lympohcytes co-cultured with adherent or suspension tumor cell types. Real time, direct analysis of T cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) using the IncuCyte Caspase-3/7 reagent. Visualize immune and tumor cell interactions. Sequential images of immune-cell killing (PBMCs) of SKOV-3 ovarian cancer cells labeled with an IncuCyte NucLight Red live cell labeling reagent.. (1) and (2) engagement of cancer cell by immune cells (3) tumor cell death, granulation & IncuCyte Caspase 3/7 green signal (4) tumor cell division. A B Quantification of trastuzumab biosimilar antibodies for antibody-dependent cell killing (ADCC). Her2 positive SKOV3 NucLight Red cells were combined with PBMCs in the presence of trastuzumab and three biosimilars. Time course and concentrationresponse curves for tumor cell number (A) and apoptotic target cell death measured using the IncuCyte Caspase-3/7 Reagent (B). No response was seen in Her2 negative cells (A549; lung carcinoma). Plate graphs for four 96-well plates provide rapid visualization of experimental results and derived Z values (C). C D 4

5 Visualize and automatically analyze phagocytic clearance of tumor cells Visualize and analyze tumor cells clearance in real-time by phagocytosis using the IncuCyte phrodo Red Cell Labeling Kit. Signal is specific to engulfed cells enabling robust measurements of antibody dependent cellular phagocytosis (ADCP) and efferocytosis. Visualize and validate phagocytic clearance of tumor cells with images and movies. J774A.1 mouse macrophages phagocytosing apoptotic Jurkats labeled using the IncuCyte phrodo Red Cell Labeling Kit over 24 hours. Fluorescent signal correlates with phenotypic changes associated with phagocytosis. IncuCyte automatically masks and quantifies fluorescent objects (blue, bottom row). Anti-CD47 antibody mediated cellular engulfment. Phagocytosis of CCRF-CEM human leukemic lymphoblasts by human bone-marrow derived macrophages. Addition of anti-cd47 binds to the don t eat me signal on CCRF-CEM to promote phagocytosis in a concentration dependent manner. 5

6 Ordering information Contact us at to place an order or for more information. APPLICATION PRODUCT QTY CAT. NO. IncuCyte ClearView 96-Well Cell Migration Plate 1 plate 4582 IncuCyte ClearView 96-well Cell Migration Plates 10 plates 4582 Chemotaxis Visualize and automatically analyze chemotactic migration IncuCyte ClearView 96-well Cell Migration Plates 50 plates 4599 IncuCyte ClearView Reservoir Plate 10 plates 4600 IncuCyte ClearView Reservoir Plates 100 plates 4601 IncuCyte Chemotaxis Software Module 1 each IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent 20 µl 4440 IncuCyte Caspase-3/7 Red Apoptosis Assay Reagent 20 µl 4704 Immune Cell Killing and Proliferation and Clustering Directly measure tumor cell killing and proliferation in real time IncuCyte Annexin V Red Reagent for apoptosis 1 vial, 100 tests 4641 IncuCyte Annexin V Green Reagent for apoptosis 1 vial, 100 tests 4642 IncuCyte NucLight Green Lentivirus Reagent (EF-1 α, Puro) 0.2mL 4624 IncuCyte NucLight Red Lentivirus Reagent (EF-1 α, Puro) 0.2mL 4625 IncuCyte CytoLight Rapid Green Reagent for cell labeling One vial, 100 1,000 tests 4705 IncuCyte CytoLight Rapid Red Reagent for cell labeling Five vials, 100 1,000 tests 4706 Phagocytosis Visualize and quantify clearance of labeled tumor cells IncuCyte phrodo Red Cell Labeling Kit 1 kit 4649 Key applications using the IncuCyte Live-Cell Analysis System Learn more at essenbioscience.com/applications Immune Cell Clustering Visualize and quantify expansion and clustering without removing your cultures from the incubator Chemotaxis Visually confirm the chemotactic migration of immune cells towards chemoattractants with ClearView 96-well plates Immune Cell Killing Detect tumor cell death directly by counting NucLight nuclear-labelled cells or measuring apoptosis with IncuCyte Caspase 3/7 reagent Phagocytosis Continuously analyze and generate movies of immune cells engulfing phrodo labelled target tumor cells 2017 Essen BioScience. All rights reserved. IncuCyte, Essen BioScience and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified C00