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

Cancer Cell Biology Cell culture Cell isolation Flow Cytometry Immunology Model Organisms Molecular Biology RNA-seq Sequencing

Journal

STAR protocols
ISSN: 2666-1667
Titre abrégé: STAR Protoc
Pays: United States
ID NLM: 101769501

Informations de publication

Date de publication:
01 Aug 2024
Historique:
received: 22 03 2024
revised: 16 05 2024
accepted: 22 06 2024
medline: 4 8 2024
pubmed: 4 8 2024
entrez: 3 8 2024
Statut: aheadofprint

Résumé

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.

Identifiants

DOI: 10.1016/j.xpro.2024.103195 PMID: 39096495
pubmed: 39096495
pii: S2666-1667(24)00360-5
doi: 10.1016/j.xpro.2024.103195
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

103195

Informations de copyright

Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests A.P., A.C., and F.A. are named inventors on patents focused on the manipulation of dendritic cells in the tumor microenvironment.

Auteurs

Athanasios Papadas (A)

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: apapadas@health.ucsd.edu.

Daniel J Lagal (DJ)

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.

Yaling Dou (Y)

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.

Duncan Hong (D)

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.

Alicia Gibbons (A)

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.

Alexander Cicala (A)

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.

Yun Huang (Y)

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.

Brolyn Zomalan (B)

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.

Elsa Molina (E)

Next Generation Sequencing Core, The Salk Institute for Biological Studies, La Jolla, CA, USA.

Fotis Asimakopoulos (F)

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: fotis@health.ucsd.edu.

Classifications MeSH