In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Published: July 31, 2023

Abstract

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. Vis. Exp. (2015)

This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with γ-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of AC-6 Feeder cells Note: The AC-6.21 (AC-6 in short) stromal cell line (provided by I. Weissman, Stanford University) should be maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS). To serve as feeder cells, AC-6 cells are γ-irradiated with …

Declarações

The authors have nothing to disclose.

Materials

Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5 Biolegend 108408 Linage marker
Anti-mouse NK1.1 (PE), clone PK136 Biolegend 108708 Linage marker
Anti-mouse CD11b (PE), cloneM1/70 Biolegend 101208 Linage marker
Anti-mouse CD19 (PE), clone eBio1D3 Biolegend 115508 Linage marker
Anti-mouse B220 (PE), clone RA3-6B2 Biolegend 103208 Linage marker/FACS
Anti-mouse CD3 (PE), clone 17A2 Biolegend 100308 Linage marker
Anti-mouse CD8a (PE), clone 53-6.7 Biolegend 100707 Linage marker
Anti-mouse MHC-II (PE), clone NIMR-4 Biolegend 107608 Linage marker
Anti-mouse Ter119 (PE), clone TER-119 Biolegend 116208 Linage marker
Anti-mouse Thy1.1 (PE), clone HIS51 eBioscience 12-0900-83 Linage marker
Anti-mouse M-CSFR (APC), clone AFS98 Biolegend 135510 FACS
Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8 Biolegend 105824 FACS
Anti-mouse Sca-1 (FITC), clone D7 Biolegend 108106 FACS
Anti-mouse IL-7Ra (PE/Cy7), clone A7R34 Biolegend 135014 FACS
Anti-mouse CD11c (PerCP/Cy5.5), clone N418 Biolegend 117328 FACS
Anti-mouse CD11b (FITC), clone M1/70 Biolegend 101206 FACS
FACSAria III BD Biosciences Cell sorter
FACS sorting tube BD Biosciences 352054
FlowJo FlowJo LLC Flow analysis sofrware

Tags

Play Video

Citar este artigo
In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors. J. Vis. Exp. (Pending Publication), e21521, doi: (2023).

View Video