Protocol for fluorescence-activated cell sorting of human EpCAM+ lung cancer cells for gene expression analysis of Rac guanine-nucleotide exchange factors

Neil T Sullivan; Mariana Cooke

doi:10.1016/j.xpro.2022.101367

Protocol for fluorescence-activated cell sorting of human EpCAM⁺ lung cancer cells for gene expression analysis of Rac guanine-nucleotide exchange factors

STAR Protoc. 2022 May 2;3(2):101367. doi: 10.1016/j.xpro.2022.101367. eCollection 2022 Jun 17.

Authors

Neil T Sullivan¹, Mariana Cooke^{2

3}

Affiliations

¹ Division of Thoracic Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
² Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
³ Department of Medicine, Einstein Medical Center Philadelphia, Philadelphia, PA 19141, USA.

Abstract

Here, we describe a protocol for fluorescence-activated cell sorting (FACS) of human EpCAM⁺ cells from fresh surgically resected specimens. We then use Q-PCR to identify specific molecular targets associated with the metastatic phenotype. This combined approach enables a qualitative and quantitative gene expression analysis of lung cancer samples. We describe how to use the protocol for Rac GEFs, but it can be applied broadly to other molecular targets. For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021) and Quatromoni et al. (2015).

Keywords: Cancer; Cell Biology; Flow Cytometry/Mass Cytometry; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Epithelial Cell Adhesion Molecule / genetics
Flow Cytometry / methods
Gene Expression
Guanine
Guanine Nucleotide Exchange Factors*
Humans
Lung Neoplasms* / genetics
Nucleotides

Substances

EPCAM protein, human
Epithelial Cell Adhesion Molecule
Guanine Nucleotide Exchange Factors
Nucleotides
Guanine

Grants and funding

T32 CA009140/CA/NCI NIH HHS/United States