A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons

Sehyoun Yoon; Peter Penzes

doi:10.1016/j.xpro.2021.101118

A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons

Sehyoun Yoon^*, Peter Penzes

^*Corresponding author for this work

Neuroscience

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to Yoon et al. (2021a).

Original language	English (US)
Article number	101118
Journal	STAR Protocols
Volume	3
Issue number	1
DOIs	https://doi.org/10.1016/j.xpro.2021.101118
State	Published - Mar 18 2022

Keywords

Cell Biology
Cell Membrane
Cell culture
Cell-based Assays
Microscopy
Molecular Biology
Molecular/Chemical Probes
Neuroscience

ASJC Scopus subject areas

Medicine(all)
Biochemistry, Genetics and Molecular Biology(all)
Immunology and Microbiology(all)
Neuroscience(all)

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10.1016/j.xpro.2021.101118

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title = "A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons",

abstract = "This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to Yoon et al. (2021a).",

keywords = "Cell Biology, Cell Membrane, Cell culture, Cell-based Assays, Microscopy, Molecular Biology, Molecular/Chemical Probes, Neuroscience",

author = "Sehyoun Yoon and Peter Penzes",

note = "Funding Information: This work was supported by NIH grants ( R01MH107182 ). Graphical abstract, Figure 1 , and Figure 8 were created with Biorender.com ( https://biorender.com/ ). Special thanks to Dr. Colleen Zaccard and Dr. Euan Parnell for proofreading. Publisher Copyright: {\textcopyright} 2021 Northwestern University",

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doi = "10.1016/j.xpro.2021.101118",

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AU - Yoon, Sehyoun

AU - Penzes, Peter

N1 - Funding Information: This work was supported by NIH grants ( R01MH107182 ). Graphical abstract, Figure 1 , and Figure 8 were created with Biorender.com ( https://biorender.com/ ). Special thanks to Dr. Colleen Zaccard and Dr. Euan Parnell for proofreading. Publisher Copyright: © 2021 Northwestern University

PY - 2022/3/18

Y1 - 2022/3/18

N2 - This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to Yoon et al. (2021a).

AB - This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to Yoon et al. (2021a).

KW - Cell Biology

KW - Cell Membrane

KW - Cell culture

KW - Cell-based Assays

KW - Microscopy

KW - Molecular Biology

KW - Molecular/Chemical Probes

KW - Neuroscience

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AN - SCOPUS:85123090123

SN - 2666-1667

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ER -

A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons

Abstract

Keywords

ASJC Scopus subject areas

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