Maintenance of Peritoneal Macrophages in the Steady State

Abstract

Resident peritoneal macrophages (M phi) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH-1. After immunofluorescence staining with M phi specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident M phi were labeled with both the green dye and red Mab label, while recruited M phi were labeled only with the red Mab tag. These populations were distinguished by two-color flow cytometry. PKH-1 labeled resident peritoneal M phi were followed for 1-49 days in mice that received no further treatment (steady state). Dye labeled M phi were still detectable after 49 days in vivo, although their green fluorescence intensity had decreased steadily over time. The decrease in dye intensity was limited to M phi, as the fluorescence intensity of PKH-1 labeled peritoneal lymphocytes did not change. Resident M phi populations were clearly separated from recruited M phi by the intensity of their staining with PKH-1 for up to 28 days. No decrease in the number of resident (dye labeled) peritoneal M phi was observed over 1-28 days. These data indicate that resident peritoneal M phi were not replaced by recruited blood monocytes in the steady state.

Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation

“…The ability to accurately identify a cellular population of interest in a heterogeneous biological environment has accelerated progress in transplantation medicine (Chawla et al, 2006;Morimoto et al, 2005;Sugito et al, 2004), cellular biology (Batard et al, 2000;Hasbold et al, 1999) and immunology (Maus et al, 2001;Melnicoff et al, 1988;Rimaniol et al, 2003;Svane et al, 2002). For nearly two decades, researchers have capitalized upon the properties of CFSE and the PKH dyes for a variety of cell tracking applications including the detection of in vivo labeled targets in both migration and functional assays, studies of the interaction of leukocytes with their cognate cellular partners, and also the analysis of isolated cellular subsets; either as an homogenous culture or in a heterogeneous cellular milieu.…”

“…This characteristic permits a labeled cell to be tracked for extended periods of time (Wallace et al, 1993) and can also be exploited for measuring proliferation, due to the halving of fluorescence intensity as daughter cells are spawned. Indeed, the CFSE and PKH dyes have been invaluable tools in the evolution of cellular studies, allowing for the measurement of cytotoxic effector activity (Lee-MacAry et al, 2001;Simmons et al, 2005;Slezak et al, 1989), monitoring cell trafficking in vivo and in vitro (Chawla et al, 2006;Horan et al, 1989;Morimoto et al, 2005;Sugito et al, 2004;Wallace et al, 1993) and studying resident versus recruited cells in the immune response (Clay et al, 2005;Maus et al, 2001;Melnicoff et al, 1988). Perhaps the most common application of such dyes is the detection of differential proliferation responses among lymphocyte subsets.…”

Ageing and the immune system: focus on macrophages

“…It is now thought that high doses of irradiation or corticosteroids could have interfered with results from initial studies. Other researchers have reported that tissue-resident macrophages are not replaced by monocytes in the steady state [52,53]. This has been verifi ed for microglia, CNS-resident macrophages, which originate from yolk sac-derived primitive myeloid progenitors and are maintained by self-renewal [54].…”

Topical Implantation of Mesenchymal Stem Cells Has Beneficial Effects on Healing of Experimental Colitis in Rats

“…Because the effect of 1 ϫ 10 6 cells was modest, for the primary implantation we used 1 ϫ 10 7 bone marrow-derived cells. Before implantation, the cells were labeled with a fluorescent cell linker, PKH67 (SigmaAldrich) (Melnicoff et al, 1988), to enable tracing the cells in vivo. The PKH67-labeling procedure for adult somatic stem cells has been described previously in detail (Askenasy and Farkas, 2002;Grøgaard et al, 2007).…”