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I N C U C Y T E ® L I V E - C E L L A N A LY S I S S Y S T E M Neuronal Cell Health Assays Real-time automated measurements of neuronal cell viability and neurite dynamics inside your incubator. I N C U C Y T E ® A S S AY S F O R N E U R O N A L C E L L H E A LT H Know the whole story – Real-time neuronal cell viability and neurite outgrowth analysis inside your incubator IncuCyte® Neuronal Cell Health Assays enable automated, real-time multiplexed analysis of neurite dynamics and cell viability. Combine label-free readouts with non-perturbing live-cell reagents to simultaneously distinguish treatments that affect one or more aspects of neuronal cell health. Get the answers you need Save time and effort Protect your cells • Never miss a data point with real-time continuous analysis. • Set up, walk away and receive unbiased, automated analysis. • Robust, reproducible assay formats suitable for screening and profiling. • Run experiments in six 96 or 384-well plates at one time. Ask more questions • Analyze stationary cultures within the stable environment of an incubator - ideal for studies over days or weeks. • Maintain optimal culture conditions with label-free analysis options and nonperturbing reagent formulations. • Compatible with a wide range of neurons (primary, stem cell derived, cell lines) in monoculture or in co-culture with astrocytes. • Multiplex neurite outgrowth analysis with cell health reagents for additional insight. Confidently assess and measure neurite outgrowth and cell viability • Rank compounds in drug discovery screens. • Investigate the cellular changes that underlie neurodegeneration and neuronal injury. • Assess factors affecting neural stem cell differentiation. Measurements of neurite outgrowth and cell viability are central to the study of drug candidates, culture conditions and the impact of environmental factors on neuronal cell health and function. Both neurite outgrowth and viability assays are routinely used in drug discovery screens and may be used to identify treatments that elicit neuroprotective effects but do not induce neurotoxicity. Neurite analysis Cytotoxicity assays Apoptosis assays Measure neurite dynamics automatically and in real-time in neuronal monocultures and cocultures with astrocytes. Real-time measurements of cell viability using simple mix-and-read protocols suitable for screening. Detect apoptosis in living cells and in real time using simple mix-and-read protocols. Learn more at www.essenbioscience.com/NeuroCellHealth 2 I N C U C Y T E ® A S S AY S F O R N E U R O N A L C E L L H E A LT H Real-time, long-term neurite analysis • Visualize and analyze long-term changes in neurite length and branch points with the IncuCyte® live cell analysis system and NeuroTrack™ software. Day 0 Day 3 Day 5 • Study label-free monocultures, or use IncuCyte® NeuroLight™ reagents or NeuroPrime™ cell kits for selective analysis of neurite dynamics in co-cultures. Neurite length: 11.2 mm/mm² Cell body cluster: 134/mm² Branch points: 259.9/mm² IncuCyte® NeuroTrack™ Software Neurite length: 55.1 mm/mm² Cell body cluster: 130/mm² Branch points: 1368/mm² Neurite length: 75.1 mm/mm² Cell body cluster: 139/mm² Branch points: 2048/mm² Label-free analysis of neurons in mono-culture Perform label-free analysis of neurons in mono-culture or use IncuCyte® Neurolight™ reagents or NeuroPrime™ cell kits to study co-cultures. IncuCyte® NeuroTrack™ Software automatically analyzes images to provide measurement of neurite length, branch points and cell body clusters. Neuron specific live-cell labeling reagents and cell kits – analyze neurite dynamics in co-cultures • NeuroLight™ Red lentivirus reagent for efficient labeling of neurons without perturbing the underlying biology. • NeuroPrime™ Cell Kit for monitoring neurite dynamics in co-cultures of primary rat neurons and astrocytes. Fluorescence analysis of neurons in co-culture with astrocytes Day 3 IncuCyte® NeuroPrime™ Cell Kit Day 10 NeuroLight™ Red lentivirus reagent can be used to transduce your choice of neuronal cell types including cell lines, primary and stem cell-derived cultures. The NeuroLight™ Red lentivirus encodes a red fluorescent protein driven off a synapsin promoter to strengthen neuronal expression and minimize non-neuronal crossover. NeuroPrime Cell Kit: Cryopreserved vials of rat forebrain neurons and astrocytes and one vial of NeuroLight Red Lentivirus reagent. The kit includes a validated protocol for optimal neuron labeling and assay performance. Ideal for studies of neurotoxicity or the biology of established neural networks. Learn more at www.essenbioscience.com/NeuroCellHealth 3 I N C U C Y T E ® A S S AY S F O R N E U R O N A L C E L L H E A LT H Visualize and quantify cell viability in real time –multiplex with neurite analysis • IncuCyte® mix-and-read reagents for continuous monitoring of cytotoxicity and apoptosis in neuronal cultures. • Simultaneous measurements of neurite outgrowth and neuronal cell health. Cytotox Red reagent Annexin V Green reagent Rat forebrain neurons 333 µM glutamate, 3 days after treatment. Rat forebrain neurons in culture with astrocytes. 300 µM glutamate (left) vs. control (right), 5 hours after treatment. Use IncuCyte® Cytotox and Annexin V reagents to positively mark and quantify apoptotic or cytotoxic cells. IncuCyte® Cytotox Red reagent labels dead rat forebrain neurons treated with 333 µM glutamate. IncuCyte® Annexin V Green reagent identifies apoptotic cells in a rat forebrain neuron and astrocyte co-culture in the presence and absence of 300 µM glutamate. Neurons are selectively labeled with the IncuCyte® NeuroLight Red reagent. Multiplexed measurements of cell viability and neurite dynamics can be made in real time. Automatically generate time courses and reveal concentrationdependent responses • Automatically generate metrics for 96- or 384-well plates. • Correlate neurite dynamics with measures of neuronal cell health. Quantify treatment effects automatically and non-invasively. Every well of a 96- or 384-well plate is imaged and analyzed automatically to provide a microplate readout of cytotoxicity over time (right). Time-courses reveal concentration-dependent treatment effects on neurite dynamics and neuronal cell health. Transform data into concentration-response curves to compare pharmacology (below). 1 40 16 N eu ri te Lengt h A nn ex in V I C 5 0 = 55 µ M E C 5 0 = 59 µ M 16 1 00 0 µ M 1 20 1 00 µ M 4 µM 1 µM 8 4 0 0 12 2 N eu ri te Le ng th (mm /m m ) 10 µM 5 Time (h ) 10 15 Learn more at www.essenbioscience.com/NeuroCellHealth 1 00 8 80 4 60 40 A nn ex in V Rea gent 40 µM G re en O bj ect C onfl ue nc e A nn ex in V Rea gent G re en O bj ect C onfl ue nc e 3 00 µ M 12 -6 -5 -4 -3 0 Log [ Glut amate] ( M) 4 I N C U C Y T E ® A S S AY S F O R N E U R O N A L C E L L H E A LT H Quick Guides Neuronal mono-cultures – label free protocol overview 1 PLATE NEURONS 2 Coat wells and seed neuronal cells. ADD TREATMENTS 3 Treat cells with neurite modulating test agents. LIVE CELL ANALYSIS Monitor every well in the IncuCyte® ZOOM live-cell analysis system. Neuronal co-cultures – fluorescence protocol overview 1 PLATE NEURONS AND TRANSDUCE Coat wells and seed neurons. Transduce neurons with NeuroLight™ Red Lentivirus Reagent. 2 PLATE ASTROCYTES & MONITOR NEUROLIGHT EXPRESSION Remove the reagent from the neurons and seed astrocytes. Monitor NeuroLight™ expression in the IncuCyte® live-cell analysis system. 3 ADD TREATMENTS When NeuroLight™ expression is maximal, treat cells with neurite modulating test agents. 4 LIVE CELL FLUORESCENT ANALYSIS Monitor every well in the IncuCyte® ZOOM live-cell analysis system. Assays compatible with your choice of neuronal cultures • The IncuCyte® system provides the flexibility to work with your choice of cells and cell models. Primary neurons Stem-cell derived neurons Neuronal-like cells Learn more at www.essenbioscience.com/NeuroCellHealth Neuronal monocultures or co-cultures 5 I N C U C Y T E ® A S S AY S F O R N E U R O N A L C E L L H E A LT H Easy and complete cell health workflow with the IncuCyte® system Simple sample preparation Set up & walk away Measure long term Analyze in real-time 96/384-well protocols— no washing, no fixing, no lifting. Setup automated acquisition and analysis parameters with confidence—no need to pre-define the assay endpoint. Flexibility to measure from hours to weeks. Make decisions about your experiment as the story unfolds. See what else you can do with the IncuCyte® System Learn more at www.essenbioscience.com/applications Apoptosis Immune Cell Killing Detect apoptosis in living cells and in real time using simple mixand-read protocols. Detect tumor cell death directly by counting NucLight™ nuclearlabeled cells or measuring apoptosis with IncuCyte® Caspase 3/7 reagent. Cytotoxicity Chemotaxis Measure real-time cell viability with simple mix-and-read protocols suitable for screening. Visually confirm the chemotactic migration of immune cells towards chemoattractants with ClearView 96-well plates. Proliferation Scratch Wound Migration and Invasion Automatically measure label-free growth or count living cells with NucLight™ nuclear labeling in real time. Investigate treatment effects on neuronal cell migration across a 2D substrate or invasion through a 3D gel matrix. Immune Cell Clustering Phagocytosis Visualize and quantify expansion and clustering without removing your cultures from the incubator. Continuously analyze and generate movies of immune cells engulfing pHrodo® labeled target tumor cells. Ordering information Contact us at [email protected] to place an order or for more information. APPLICATION PRODUCTS Neurite analysis Measure neurite dynamics in monocultures and co-cultures with astrocytes. Apoptosis Detect and quantify apoptotic cells in real time. Reagents Software Reagents IncuCyte® NeuroLight™ Red IncuCyte® NeuroPrime™ Cell Kit Detect and count non-viable cells in real time. Cell lines Cryopreserved neuronal cells and astrocytes. Reagents Neuronal Cells CAT. NO. 0.45 mL 4584 1 Kit 4585 IncuCyte NeuroTrack Software 9600-0010 IncuCyte® Annexin V Red for Apoptosis 1 vial, 100 tests 4641 IncuCyte® Annexin V Green for Apoptosis 1 vial, 100 tests 4642 20 μL 4440 IncuCyte® Cytotox Red 5 μL x 5 4632 IncuCyte® Cytotox Green 5 μL x 5 4633 Neuro-2a NucLight™ Green Cells 2 x 10 cells/vial 4511 Neuro-2a NucLight™ Red Cells 2 x 10 cells/vial 4512 NeuroPrime™ rAstrocytes 2 x 10 cells/vial 4586 NeuroPrime™ rForebrain Neurons 2 x 10 cells/vial 4587 Caspase-3/7 Cytotoxicity QUANTITY 6 6 6 6 Learn more at www.essenbioscience.com/NeuroCellHealth © 2016 Essen BioScience. All rights reserved. All trademarks are the property of Essen BioScience unless otherwise specified. 8000-0482-A00