Isolation of Human Conjunctival Mast Cells and Epithelial Cells: Tumor Necrosis Factor-a from Mast Cells Affects Intercellular Adhesion Molecule

METHODS. Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-a (TNFa) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNFa protein. An examination for intercellular adhesion molecule 1 OCAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNFa supernatants from calcium ionophore A23187 (Cal)-stimulated mast cells or appropriate controls using FACS analysis.