In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

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 …

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