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SUMMARY Electrophysiology offers a high-resolution method for real-time measurement of neural activity. Longitudinal recordings from high-density microelectrode arrays (HD-MEAs) can be of considerable size for local storage and of substantial complexity for extracting neural features and network dynamics. Analysis is often demanding due to the need for multiple software tools with different runtime dependencies. To address these challenges, we developed an open-source cloud-based pipeline to store, analyze, and visualize neuronal electrophysiology recordings from HD-MEAs. This pipeline is dependency agnostic by utilizing cloud storage, cloud computing resources, and an Internet of Things messaging protocol. We containerized the services and algorithms to serve as scalable and flexible building blocks within the pipeline. In this paper, we applied this pipeline on two types of cultures, cortical organoids andex vivobrain slice recordings to show that this pipeline simplifies the data analysis process and facilitates understanding neuronal activity.
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Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence
During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co‐localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA‐seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi‐well slides are included.
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- Award ID(s):
- 2033654
- PAR ID:
- 10562462
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 3
- Issue:
- 10
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/cpz1.915
