Protocol for electrical modulation of ERK signaling dynamics in cell lines

• Published in: STAR protocols Vol. 3; no. 4; p. 101752
• Main Authors: Zhu, Kan, Guo, Liang, Patel, Preena, Albeck, John, Qing, Quan, Losert, Wolfgang, Zhao, Min
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2022; Elsevier
• Subjects: Biotechnology and bioengineering; Cell biology; Cell culture; Cell Line; Extracellular Signal-Regulated MAP Kinases - metabolism; MAP Kinase Signaling System; Microscopy; Phosphorylation; Protocol; Signal Transduction; Cell biology; Cell culture; Signal transduction; Microscopy; Biotechnology and bioengineering
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101752

Here, we describe a protocol for modulating the dynamics of the extracellular signal-regulated kinases (ERK) pathway in a customized alternating current (AC) electric field stimulation chamber. We use an ERK translocation reporter that can accurately represent the intracellular ERK activity in real time without chemical agents or gene disruption. ERK activation is assessed by comparing the relative intensity of nuclear fluorescence to cytosolic fluorescence in live-cell conditions. The approach can be applied to other signaling pathways as well. For complete details on the use and execution of this protocol, please refer to Guo et al. (2021). [Display omitted] •Protocol for robust electrical activation of intracellular signaling pathways•Step-by-step guide for delivering AC electric field in a customized chamber•Rapid quantification of single-cell ERK dynamics in time-lapse fluorescence images•Precise control of ERK dynamics by stimulation with different scheme Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we describe a protocol for modulating the dynamics of the extracellular signal-regulated kinases (ERK) pathway in a customized alternating current (AC) electric field stimulation chamber. We use an ERK translocation reporter that can accurately represent the intracellular ERK activity in real time without chemical agents or gene disruption. ERK activation is assessed by comparing the relative intensity of nuclear fluorescence to cytosolic fluorescence in live-cell conditions. The approach can be applied to other signaling pathways as well.