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In vivo labeling of resident peritoneal macrophages
M J Melnicoff1, P S Morahan, B D Jensen
1Department of Immunology, Smith, Kline and French Laboratories, Swedeland, King of Prussia, PA 19406.
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.
Insights
A novel green fluorescent dye allows researchers to label resident peritoneal macrophages (M phi) in vivo. This method distinguishes resident from recruited M phi using flow cytometry without harmful procedures.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Peritoneal macrophages (M phi) play crucial roles in immunity and tissue homeostasis.
- Distinguishing resident from recruited M phi is essential for understanding immune responses.
- Current methods for M phi labeling often involve radioisotopes or bone marrow ablation, which have limitations.
Purpose of the Study:
- To develop a novel, non-invasive method for labeling resident peritoneal macrophages (M phi).
- To enable differentiation between resident and recruited M phi populations in vivo.
- To assess the safety and efficacy of the new M phi labeling technique.
Main Methods:
- Intraperitoneal injection of a green fluorescent dye for selective M phi uptake.
- Cell surface antigen labeling using monoclonal antibodies (Mabs) and phycoerythrin-conjugated secondary antibodies.
- Two-color flow cytometry to distinguish dye-labeled resident M phi from Mabs-labeled recruited cells.
Main Results:
- The green dye selectively labeled resident M phi, which could be co-labeled with red Mabs (F4/80 or Mac 1).
- Recruited M phi and neutrophils were only labeled by the red Mabs, allowing clear distinction via flow cytometry.
- No adverse effects on cell surface antigen expression or M phi cytotoxicity were observed.
- The labeling method induced mild peritoneal inflammation but did not interfere with cell analysis.
Conclusions:
- This novel dye-based method provides an effective and safe way to label resident peritoneal macrophages (M phi) in vivo.
- The technique allows for reliable differentiation of resident and recruited M phi populations without radioisotopes or irradiation.
- This approach facilitates detailed analysis of M phi populations in individual animals for immunological studies.