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 …
開示
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 |
