The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.
Alternative protocol to obtain thioglycollate elicited macrophages:
This method is used to obtain a higher yield of macrophages.
Compared to the nonelicited approach, approximately 10 times more macrophages can be collected. The only concern is that macrophages obtained by this procedure differ in some of their physiological properties.
Representative results:
From an unmanipulated mouse, 5-10 million peritoneal cavity cells can be obtained with a good isolation protocol. Among all live cells, 50-60% are B cells, ~30% macrophages and 5-10% are T cells (Fig 1).
Figure 1. Phenotype of cells isolated from the peritoneal cavity. Following isolation of peritoneal cavity cells they were stained with anti-mouse TCRβ-FITC, B220-PE-Texas red, CD11b-Pacific blue, CD23-PE-Cy7 and CD5-APC. Representative flow cytometric plots show percentages of B and T cells (a), macrophages (b), B1 and B2 cells (c) and B1a and B1b cells (d).
Isolation of peritoneal cavity cells is an important technique for the study of different immune cells, primarily macrophages and specific B cell subsets. Although this is a simple process, there are some critical steps involved. Presence of contaminating blood in the collected sample should be avoided to obtain a pure peritoneal cavity cell population. Euthanasia by cervical dislocation should be done carefully to avoid blood contamination in the peritoneal cavity. Alternatively CO2 can be used. In addition, during the procedure proper care should be taken to avoid puncturing the bladder or any other organs in the peritoneal cavity. Recovering most of the injected fluid is also important to the cell yield.
This procedure is widely used to study macrophage biology since moderate numbers of resident, unstimulated macrophages can easily be obtained from the peritoneal cavity, in contrast to the laborious task of differentiating myeloid progenitor cells into mature macrophages in vitro, using macrophage colony stimulating factor (8,9). The macrophage yield from the peritoneal cavity can be improved by using the thioglycollate elicitation method, although thioglycollate use might alter physiological properties of the macrophages (7).
B cells are an important part of our immune system playing crucial roles in both innate and adaptive immunity. Among different B cell subpopulations, B1 cells comprise a unique subset of B cells involved in innate immunity, autoimmunity and immune regulation. B1 cells are primarily located in the peritoneal cavity and are self replenishing. They are one of the primary producers of natural IgM which provides the first line of protection against a number of virus and bacteria (10-12). B1 cells are also a major source of IL-10 (6), which is an important cytokine involved in immune regulation. Although several studies have been pursued with the B1 cells, still there is scope for further evaluation of their contrasting functions, specially their regulatory role. Peritoneal cavity cell isolation method provides the unique opportunity to study the functions of B1 cells.
This work was supported in part by NIH grant AI069358 and the BloodCenter Research Foundation.
Material Name | Tip | Company | Catalogue Number | Comment |
---|---|---|---|---|
Thioglycollate Medium, Brewer Modified | BD BBL | 211716 |