Protocol to identify and isolate rare murine tumor-resident dendritic cell populations for low-input transcriptomic profiling

Athanasios Papadas; Daniel J Lagal; Yaling Dou; Duncan Hong; Alicia Gibbons; Alexander Cicala; Yun Huang; Brolyn Zomalan; Elsa Molina; Fotis Asimakopoulos

doi:10.1016/j.xpro.2024.103195

Protocol to identify and isolate rare murine tumor-resident dendritic cell populations for low-input transcriptomic profiling

STAR Protoc. 2024 Sep 20;5(3):103195. doi: 10.1016/j.xpro.2024.103195. Epub 2024 Aug 1.

Authors

Affiliations

¹ Division of Blood and Marrow Transplantation, Department of Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA; Moores Cancer Center, University of California, San Diego (UCSD), La Jolla, CA, USA. Electronic address: [email protected].
² Division of Blood and Marrow Transplantation, Department of Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA; Moores Cancer Center, University of California, San Diego (UCSD), La Jolla, CA, USA.
³ Next Generation Sequencing Core, The Salk Institute for Biological Studies, La Jolla, CA, USA.
⁴ Division of Blood and Marrow Transplantation, Department of Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA; Moores Cancer Center, University of California, San Diego (UCSD), La Jolla, CA, USA. Electronic address: [email protected].

Abstract

Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.¹.

Keywords: Cancer; Cell Biology; Cell culture; Cell isolation; Flow Cytometry; Immunology; Model Organisms; Molecular Biology; RNA-seq; Sequencing.

MeSH terms

Animals
Cell Separation / methods
Dendritic Cells* / cytology
Dendritic Cells* / metabolism
Flow Cytometry / methods
Gene Expression Profiling* / methods
Mice
Mice, Inbred C57BL
Neoplasms / genetics
Neoplasms / immunology
Neoplasms / pathology
Transcriptome / genetics
Tumor Microenvironment / genetics

Abstract

MeSH terms

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