Flow Cytometric Analysis of Dendritic Cell Markers

Formalin-fixed and fresh tissues were cut into sections, rubbed gently with plastic forceps and rinsed with physiological saline. The suspension was filtered with a 300-mesh copper grid to remove the block, and the cell suspension was centrifuged at 150 × g for 2 min. The cells (1×10⁶/ml) were collected and washed twice in PBS. A flow cytometer (Epics-XL II; Beckman Coulter, Inc.) was used to determine the cell surface expression of CD1a, CD83, CD86 and CD80 according to previous protocol (13 (link),14 (link)). Briefly, mononuclear cells were stained using monoclonal antibodies to CD1a, CD83, CD86, CD80 (Beckman Coulter, Inc., Brea, CA, USA) and appropriate IgG isotype controls. Cells were kept at 4°C for 30 min, and filtered using a 500-mesh copper grid. The samples were analyzed using a FACSCalibur cytometer and CellQuest Pro software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).