Neuronal Cell Health Assays. Real-time automated measurements of neuronal cell viability and neurite dynamics inside your incubator.

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1 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.

2 See what your cells are doing and when they do it Real-time visualization and analysis of cell motility 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. Ask new questions Get new answers Improve productivity Protect your cells Study a range of neuronal cell types primary, stem cell-derived, cell lines in monoculture or in co-culture with astrocytes. Perform screening and profiling with robust, reproducible assay formats. Never miss a data point with real-time continuous analysis. Multiplex neurite outgrowth analysis with cell health reagents for additional insight. Enjoy walk-away convenience as images are automatically acquired and analyzed. Run experiments in up to six 96- or 384-well plates simultaneously. Perform analysis without ever removing cells from the incubator or disturbing cultures. Maintain cell health with non-perturbing, mix-and-read reagent formulations. 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 Measure neurite dynamics automatically and in real-time in neuronal monocultures and co-cultures with astrocytes. Cytotoxicity assays Real-time measurements of cell viability using simple mix-and-read protocols suitable for screening. Apoptosis assays Detect apoptosis in living cells and in real time using simple mix-and-read protocols. 2

3 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. Study label-free monocultures, or use IncuCyte NeuroLight reagents or NeuroPrime cell kits for selective analysis of neurite dynamics in co-cultures. Day 0 Day 3 Day 5 Neurite length: 11.2 mm/mm² Cell body cluster: 134/mm² Branch points: 259.9/mm² 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 IncuCyte NeuroTrack Software 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 Day 10 IncuCyte NeuroPrime Cell Kit 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. 3

4 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 Rat forebrain neurons 333 µm glutamate, 3 days after treatment. Annexin V Green reagent 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 concentration-dependent 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 concentrationdependent treatment effects on neurite dynamics and neuronal cell health. Transform data into concentrationresponse curves to compare pharmacology (below) N eurite Length I C 5 0 = 55 µ M Annexin V E C 5 0 = 59 µ M µ M A nn ex in V Rea gent G re en O bj ect C onfl ue nce µ M 1 00 µ M 4 0 µ M 1 0 µ M 4 µ M 1 µ M N eu ri te Leng th (mm /m m 2 ) A nn ex in V Rea gent G re en O bj ect C onfl ue nce Time (h ) Log [Glutamate] (M) 4 0

5 Quick Guides Neuronal mono-cultures label free protocol overview 1 PLATE NEURONS 2 ADD TREATMENTS 3 LIVE CELL ANALYSIS Coat wells and seed neuronal cells. Treat cells with neurite modulating test agents. Monitor every well in the IncuCyte live-cell analysis system. Neuronal co-cultures fluorescence protocol overview 1 PLATE NEURONS AND TRANSDUCE 2 PLATE ASTROCYTES & MONITOR NEUROLIGHT EXPRESSION 3 ADD TREATMENTS 4 LIVE CELL FLUORESCENT ANALYSIS Coat wells and seed neurons. Transduce neurons with NeuroLight Red Lentivirus Reagent. Remove the reagent from the neurons and seed astrocytes. Monitor NeuroLight expression in the IncuCyte live-cell analysis system. When NeuroLight expression is maximal, treat cells with neurite modulating test agents. Monitor every well in the IncuCyte 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 Neuronal monocultures or co-cultures 5

6 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. Ordering information Contact us at to place an order or for more information. APPLICATION PRODUCTS QUANTITY CAT. NO. Neurite analysis Measure neurite dynamics in monocultures and co-cultures with astrocytes. Apoptosis Detect and quantify apoptotic cells in real time. Cytotoxicity Detect and count non-viable cells in real time. Cell lines Cryopreserved neuronal cells and astrocytes. Reagents IncuCyte NeuroLight Red 0.45 ml 4584 IncuCyte NeuroPrime Cell Kit 1 Kit 4585 Software IncuCyte NeuroTrack Software Reagents Reagents Neuronal Cells IncuCyte Annexin V Red for Apoptosis 1 vial, 100 tests 4641 IncuCyte Annexin V Green for Apoptosis 1 vial, 100 tests 4642 Caspase-3/7 20 μl 4440 IncuCyte Cytotox Red 5 μl x IncuCyte Cytotox Green 5 μl x Neuro-2a NucLight Green Cells 2 x 10 6 cells/vial 4511 Neuro-2a NucLight Red Cells 2 x 10 6 cells/vial 4512 NeuroPrime rastrocytes 2 x 10 6 cells/vial 4586 NeuroPrime rforebrain Neurons 2 x 10 6 cells/vial 4587 See what else you can do with the IncuCyte Live-Cell Analysis System Learn more at Apoptosis Detect apoptosis in living cells and in real time using simple mix-and-read protocols. Cytotoxicity Measure real-time cell viability with simple mix-and-read protocols suitable for screening. Immune Cell Killing Detect tumor cell death directly by counting NucLight nuclear-labeled cells or measuring apoptosis with IncuCyte Caspase 3/7 reagent. Chemotaxis Visually confirm the chemotactic migration of immune cells towards chemoattractants with ClearView 96-well plates. Proliferation Automatically measure label-free growth or count living cells with NucLight nuclear labeling in real time. Immune Cell Clustering Visualize and quantify expansion and clustering without removing your cultures from the incubator. Scratch Wound Migration and Invasion Characterize the metastatic potential of tumor cells and investigate treatment effects on migration across a 2D substrate or invasion through a 3D gel matrix. Phagocytosis Continuously analyze and generate movies of immune cells engulfing phrodo labeled target tumor cells 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 B00