Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures

Cells produced in spleen stroma-dependent long-term cultures (LTC) have now been clearly defined as dendritic cells (DC). Characterization of cells by antibody staining and FACS analysis has only been possible using a procedure to quench the high autofluorescence of DC produced in LTC (LTC-DC). The population of large cells produced by the established LTC-X1 culture are homogeneously positive for a number of cell-surface markers expressed by DC. These include CD11c, CD11b, Dec-205, Fc receptor and CD86. They also express markers detectable with the F4/80 and 33D1 antibodies. Cells produced in LTC do not uniformly express the MHC II marker, consistent with an immature DC phenotype. Most cells are weakly positive for MHC II with a small subset of highly positive cells. The quenching method involves staining cells with crystal violet dye, which is taken up within the cell. The importance of optimizimg fluorescent antibody staining assays for delineating DC subsets is indicated and the LTC system is established as a valuable and continuous source of DC precursors