Live‐Cell Imaging and Analysis with Multiple Genetically Encoded Reporters

Abstract

Genetically encoded live-cell reporters measure signaling pathway activity at the cellular level with high temporal resolution, often revealing a high degree of cell-to-cell heterogeneity. By using multiple spectrally distinct reporters within the same cell, signal transmission from one node to another within a signaling pathway can be analyzed to quantify factors such as signaling efficiency and delay. With other reporter configurations, correlation between different signaling pathways can be quantified. Such analyses can be used to establish the mechanisms and consequences of cell-to-cell heterogeneity and can inform new models of the functional properties of signaling pathways. In this unit, we describe an approach for designing and executing live-cell multiplexed reporter experiments. We also describe approaches for analyzing the resulting time-course data to quantify correlations and trends between the measured parameters at the single-cell level. © 2018 by John Wiley & Sons, Inc.

Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells

“…All data processing was performed in MATLAB using previously described methods (Gillies et al, 2020;Pargett and Albeck, 2018;Pargett et al, 2017). MATLAB scripts will be provided upon request by the Lead Contact.…”

Signalling dynamics, cell decisions, and homeostatic control in health and disease

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Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity

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