In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors
Transcrição
Plasmacytoid dendritic cells, or pDCs, are immune cells that respond to infection by producing the cytokine type-I interferon.
To generate pDCs in vitro, take a suspension of common lymphoid progenitor cells, or CLPs, isolated from mouse bone marrow. CLPs can differentiate into lymphocytes and dendritic cells.
Add the cell suspension over a layer of feeder cells — adherent stromal cells — irradiated with gamma rays to inhibit proliferation. The feeder cells provide cytokines and cell-cell contact required for the differentiation of CLPs.
Add an adequate concentration of Fms-like tyrosine kinase 3 ligand, or FL — a bivalent cytokine that promotes pDC development — and incubate.
The CLPs migrate between the feeder cells and proliferate to form groups of cells, termed cobblestone areas.
The FL in the medium binds to the Flt3 receptor — a tyrosine kinase receptor — on the CLPs. The binding induces receptor dimerization and the autophosphorylation of the tyrosine residues on the cytoplasmic domain.
Cytoplasmic adaptor proteins bind to these residues and become phosphorylated, transducing the signal to the downstream signaling components. The signaling cascade causes the differentiation of CLPs into pDCs — small round cells that grow in suspension — as well as conventional dendritic cells, or cDCs — larger spindle-shaped adherent cells.
Collect the cells. Add antibodies against pDC- and cDC-specific surface markers. Analyze the cells using flow cytometry.
Cells positive for pDC-specific markers and negative for cDC-specific markers indicate successful pDC development.
