The generation and maturation of neuronal networks are necessary characteristics of neurogenesis, providing confirmation that the cellular processes required for proper neuronal function have been developed. Recent advances in human induced pluripotent stem cell (hiPSC)-derived neurons offer promise as a powerful in vitro model strategy for the study of both the basic mechanisms of development as well as complex disorders of the human nervous system. A wide array of research areas, including molecular and developmental neuroscience, drug development and neurotoxicology, employ hiPSC-derived cell models to gain functional insight into relevant neuronal networks and disease states. However, characterization and optimization of these models as they differentiate, mature, and become functionally active are limited by current techniques.
Traditional techniques for measuring activity often:
The Incucyte® Neuronal Activity Assay is an end-to-end solution consisting of instrumentation, software, and reagent that enables unprecedented access to complex, neuronal activity measurements enabling functional insight into neuronal cell models. A new era of discovery awaits....
Gain insight into the activity and connectivity of neuronal networks with continuous imaging and analysis. The Incucyte® Neuronal Activity Assay allows for direct, long-term measurements of synaptic activity for the characterization of neuronal cell models in physiologically relevant conditions. Visualize and analyze spontaneous neuronal activity for weeks or months with the Incucyte® Live-Cell Analysis System, Incucyte® Neuronal Activity Analysis Software, and Incucyte® Neuroburst Orange Lentivirus.
Express a genetically-encoded fluorescent calcium sensor using Incucyte® Neuroburst Orange Lentivirus in a variety of neuronal cell types
Automatically capture and analyze short-term, calcium-flux kinetics from thousands of cells while qualitatively monitoring cell morphology
Quantify longitudinal changes for every active object within every well of a 96-well plate while cells sit undisturbed inside your incubator
Gain unprecedented access to phenotypic information using a lab-tested live-cell reagent, established protocol and purpose-built software – enabling even first time users
Figure 1. Efficient, non-perturbing labeling of living cells enables chronic, long-term neuronal activity analysis during network development. CNS.4U iPSC-derived neurons (Ncardia) seeded at 20,000 cells/well in a 96-well microplate were infected with Incucyte® Neuroburst Orange Reagent to monitor neuronal activity over time. Movies (3 minute duration) captured on Day 15 and day 44 reveal spontaneous development of neuronal activity (calcium oscillations) while phase images enable qualitative inspection of cell morphology. Movies were taken for 3 minutes every 24 hours over the course of 45 days, reveal maintained expression of Incucyte® Neuroburst Orange Reagent. Quantification of the orange fluorescent signal kinetics shows an increase in active object count over the course of the experiment along with a corresponding increase in mean correlation (connectivity) at later time points, signifying development of a functional, mature network.
Automatically capture and analyze short-term, calcium-flux kinetics for every active cell within each well of a 96-well plate using Incucyte® Stare Mode Movie acquisition. Easily generate correlation metrics to reveal the extent of connections in a network.
Figure 2. Analyze calcium dynamics for every active neuron in a 96-well plate. Incucyte® rCortical Neurons primary neurons seeded at 15,000 cells/well in a co-culture model with Incucyte® rAstrocytes were subsequently infected with the Incucyte® Neuroburst Orange Lentivirus. Vessel View (A) displays a summary of active objects (range image) in each well of a 96-well plate acquired during movie acquisition on day 8, revealing differences in activity across the microplate. Identification of each active object via masking (shown in purple) of the range image (B) is performed using the integrated Incucyte® Neuronal Activity Analysis Software Module. 96-well Summary Traces for movies acquired at a given timepoint (C) provide a full 96-well view of burst intensity, active object count (left inset value) and mean correlation (right inset value).
Figure 3. Identify changes in activity patterns over weeks or months to characterize neuronal cell models. iCell GlutaNeurons (Cellular Dynamics International) were seeded at 30K cells/well with a co-culture of rat astrocytes (15K cells/well) on PEI/laminin coated 96-well culture plates. Neurons were transduced with Incucyte® Neuroburst Orange Lentivirus at DIV 2, and spontaneous neuronal activity was analyzed over a period of 20 days. Kinetic quantification (center graph) of longitudinal, dynamic changes in neuronal activity of mean burst rate and mean correlation over time shows that during neuronal network maturation, an increase in burst rate occurs, peaking at day 5. Time course data also shows an increase in neuronal synaptic connections, as noted in an increase in correlation. Active object traces (corner traces) provide detailed insight into the dynamic changes in neuronal activity and connectivity for every acquired movie and are qualitatively confirmed with movie viewing tools.
Enable scientific discovery with an end-to-end solution – reagent, protocol, purpose-built acquisition and analysis software
Figure 4. Make the complex simple. Combine the power of automated acquisition and analysis with our novel reagent and lab-tested protocol to gain unprecedented access to phenotypic information. Guided interface enables rapid experimental set up and analysis, making data and images readily accessible for the entire research team.
“The Incucyte Neuronal Activity Assay is particularly useful in comparing the network activity of neuronal cultures from different genotypes and we have found it indispensable for optimizing culture conditions for the generation of mature neurons from human stem cells.”
Michael Uhler, PhDb | Professor, Biological Chemistry
Research Professor, Molecular and Behavioral Neuroscience Institute, University of Michigan
“The Incucyte Neuronal Activity Assay allows for testing multiple culture conditions across multiple neuronal lines. The assay let us assess the impact of co-culture between different cell types, multiplexed with different treatment conditions at a semi high-throughput scale in a manner previously not possible."
Krishna Vadodaria, PhD | Research Associate
Rusty Gage Lab, Salk Institute
“The Incucyte Neuronal Activity Assay allowed me to perform a longitudinal analysis of calcium transients in stem cell-derived neurons. The 96-well plate format allows multiple concurrent comparisons from different cell lines and growth conditions. The burst rate, burst duration, and network formation was highly reproducible with minimal well-to-well variability. The ability to concurrently view cell morphology with phase contrast was highly advantageous to eliminate outliers.”
Louis Dang, MD, PhD | Clinical Lecturer
Jack Parent Lab, University of Michigan