In Vivo Labeling of Resident Peritoneal Macrophages

Abstract

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. 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 M phi were labeled by both the green dye and the red Mab markers, while recruited M phi 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 M phi 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 M phi 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 M phi, 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 M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.

Impact of carbon dioxide versus air pneumoperitoneum on peritoneal cell migration and cell fate

“…By the use of a specific linker technique, dye aggregates or particulates are formed and ingested by phagocytosis in vivo or in vitro without evidence of functional impairment [16,17]. This technique allows the quantity of intravascular monocytes that have become peritoneal macrophages within a specified period to be determined [16], as well as the quantity of polymorphonuclear leukocyte granulocytes migrating from the blood compartment into the peritoneum.…”

Ex Vivo PKH26‐Labelling of Lymphocytes for Studies of Cell Migration In Vivo

“…Previously, it has been demonstrated that PKH labelled cells grow normally in culture [14], react normally in cytotoxic assays [7,14] and that the incorporation of PKH does not physically weaken the membrane of the cells [15]. However, at higher concentrations there can be difficulties in the detection of cell surface antigens using flow cytometry [6].…”

“…The newer fluorescent labelling molecule PKH, which is incorporated into the lipid bilayer of cytoplasmic membranes, has been used for labelling a variety of cells, including peripheral lymphocytes [6], macrophages [7] and erythrocytes [8].…”

Tissue Engineering for Tissue and Organ Regeneration

“…The prerequisites for using PKH26 for labelling of cells has been demonstrated in the previous study by many researchers that the incorporation of PKH dye does not physically weaken the membrane of the cells, does not interfere with the functions of the cells and appeared to be ideal for long-term tracking of cells. Animal studies revealed no toxicity of the PKH dye and no immune response appears to generate against labelled cells (Read et al, 1991;Slezak andHoran, 1989a, 1989b;Melnicoff et al, 1988). Most importantly, Johnsson et al (1997) had reported in their 'ex vivo PKH26-labelling of lymphocytes for studies of cell migration in vivo' study of this labelling dye was found to be retained within the labelled cells and not transferred to other cells.…”