Identification and Isolation of Synovial Dendritic Cells

In rheumatoid arthitis patients, three compartments need to be considered: peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST). Dendritic cells (DC) characterized from each compartment have different properties. The methods given are based on cell sorting for isolation of cells, and flow cytometry and immunohistochemical staining for analysis of cells. Myeloid non-T cells are first enriched by density gradient centrifugation, sheep erythocyte rosetting, and, in some cases, magnetic immunodepletion. By flow cytometry, DC can then be analyzed or sorted based on two- or three-color immunofluorescence. Some variations on this basic theme are also outlined. The basic protocol for two-color immunohistochemistry of formalin-fixed ST is then given. This is based on the localization of the NFκB family member, RelB, to the nucleus of differentiated DC, and exclusion of B cells, macrophages, and follicular DC by double staining. Some variations that are particularly useful in frozen sections follow.