3D time-lapse microscopy paired with endpoint lineage analysis in mouse blastocysts

Michael J Pokrass; Sergi Regot

doi:10.1016/j.xpro.2021.100446

3D time-lapse microscopy paired with endpoint lineage analysis in mouse blastocysts

STAR Protoc. 2021 Apr 8;2(2):100446. doi: 10.1016/j.xpro.2021.100446. eCollection 2021 Jun 18.

Authors

Michael J Pokrass^{1

2

3}, Sergi Regot^{1

2

3}

Affiliations

¹ Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
³ Biochemistry, Cellular, and Molecular Biology Graduate Program, Baltimore, MD, USA.

Abstract

Determining how signaling dynamics relate to gene expression and cell fate is essential to understanding multicellular development. We present a unified live imaging and lineage analysis method that allows integrated analysis of both techniques in the same mouse embryos. This protocol describes the embryo isolation, confocal imaging, immunofluorescence, and in silico alignment required to connect time-lapse and endpoint measurements. By utilizing different biosensors and fixed readouts, this method allows interrogation of signaling dynamics that specify cell fates in developing embryos. For complete details on the use and execution of this protocol, please refer to Pokrass et al. (2020).

Keywords: Cell differentiation; Microscopy; Signal transduction; Single cell.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Blastocyst* / cytology
Blastocyst* / metabolism
Blastocyst* / physiology
Cells, Cultured
Female
Imaging, Three-Dimensional
Male
Mice
Microscopy, Confocal / methods*
Molecular Probe Techniques*
Signal Transduction / physiology
Time-Lapse Imaging / methods*

Grants and funding

R35 GM133499/GM/NIGMS NIH HHS/United States