In Vivo Labeling of Resident Peritoneal Macrophages

A novel method for labeling resident peritoneal macrophages (MO) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident Mø. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident Mø were labeled by both the green dye and the red Mab markers, while recruited Mø or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two‐color flow cytometry. This technique enabled identification of resident and recruited Mø in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that Individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or Mø mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the Mø, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the Mø labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.