Name: isolation of leukocytes from the murine tissues at the maternal-fetal interface
Uploaded: 2015-05-21T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface at JoVE.com

NGL was supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. We gratefully acknowledge Maureen McGerty and Amy E. Furcron (Wayne State University) for their critical reading of the manuscript.

Before working with the samples mentioned in this protocol, animal ethical approval must be given by the Local Research Ethics Committee and the Institutional Review Boards. When working with animal blood, cells, or hazardous agents as mentioned in this protocol, the proper biosafety and laboratory safety actions must be followed.
1. Mouse Handling and Tissue Collection
<ol>
	<li>Prepare a sterile workstation and obtain sterile tools for tissue collection. These tools will include large and ...

The collection of consistent data that records the abundance and phenotypic characteristics of infiltrating leukocytes at the maternal-fetal interface is essential to understanding the pathogenesis of pregnancy-related complications. Several techniques have been described that facilitate the isolation of infiltrating leukocytes from the murine tissues at the maternal-fetal interface throughout pregnancy31,38,39,43-46. However, each technique is different, uses different enzymes or enzyme combinations, requires...

Immune tolerance in pregnancy is a period when distinctive changes occur within the immune system of the mother. These changes allow the mother to tolerate the fetus, a semi-allogenic graft1. The fetus expresses paternal major histocompatibility complex (MHC) antigens2, and fetal cells have been found in the maternal circulation3; however, the fetus is not rejected4,5. This enigma is not fully understood.
The most recent hypothesis states that maternal-fetal tolerance is created during coitus and fecundation6,7 and maintained to sustain a full-term pregnancy8-10. A breakdown of this maternal-fetal tolerance is considered a mechanism of disease during early and late stages of pregnancy10-16. Maternal-fetal tolerance involves the participation of various leukocyte sub-populations, including T cells (regulatory T cells, Th1 cells, Th2 cells, and Th17 cells), macrophages, neutrophils, mast cells, NK cells, and NKT cells, dendritic cells, and B cells, that change in density and localization throughout pregnancy15,17-19. Maternal-fetal tolerance is enriched at the maternal-fetal interface20 - the anatomical site where the immune system of the mother interacts with the fetal antigens20,21.
The maternal-fetal interface is created during placentation when the fetal extravillous trophoblast cells invade the uterine mucosa22-24. On the fetal side of this interface, the membranes surrounding the fetus create a specialized epithelial surface within the placenta, and the syncytiotrophoblast cells control the nutrient exchange through their direct contact with maternal blood22. On the maternal side of the interface, the decidua recruits a heterogeneous pool of leukocytes that in mice account for 30% to 50% of all decidual cells. In addition to their participation in maternal immune tolerance, these cells are key contributors to different processes during pregnancy, e.g., the protection of the reproductive tract from infections, fecundation, embryo implantation7,25, decidual angiogenesis26, vascular remodeling24,27, trophoblast invasion28, placental development24,25, and, ultimately, labor and delivery15,17. Therefore, the study of the leukocytes involved in maternal-fetal tolerance is essential to elucidating the pathogenesis of pregnancy-related complications.
While the use of immunohistochemistry and immunofluorescence has generated data for the direct visualization and localization of uterine, decidual, or placental leukocytes29,30, flow cytometry analysis has further revealed specific subsets of leukocytes in each of these tissues31,32. Additionally, flow cytometry has been used to determine the density and proportion of maternal-fetal interface leukocytes33 and expression levels of extracellular and intracellular proteins8-10,34. Flow cytometric analysis of leukocytes at the maternal-fetal interface requires a single-cell suspension. In order to isolate infiltrating leukocytes from the decidual, uterine, and placental tissues, two methods of tissue dissociation have been used: mechanical and enzymatic. Both methods allow the separation of infiltrated leukocytes from the extracellular matrix (ECM) of these tissues. Enzymatic tissue dissociation is superior to mechanical tissue dissociation as it allows a higher yield of leukocytes with less shear-force-associated damage35. Consequently, mechanical tissue dissociation requires pooling tissues36, which may increase the variability and heterogeneity of the samples. Yet, mechanical dissociation may be the choice when the antigen of interest can be altered by enzymatic dissociation or when the functionality of the cells of interest need to be preserved (e.g., cytotoxicity of NK cells)35.
The use of proteolysis with specific enzymes to degrade the ECM eliminates the low yields observed with mechanical dissociation. Several studies have reported the use of trypsin32, collagenase37, DNase31, dispase38, and commercial cocktails of various enzymes32,39. However, the nature and concentration of different enzymes and the duration of digestion must be meticulously defined and validated in order to ensure maintenance of the integrity of the cell surface antigenic epitopes required for immunophenotyping. The various surface structures are differentially susceptible to destruction by different enzymes, with some enzymes, such as trypsin, being notorious for stripping leukocyte surface epitopes recognized by many monoclonal antibodies.
Introduced herein is a method using a proteolytic and collagenolytic enzymatic cocktail, called Accutase. This enzymatic solution is gentle enough while still efficient in dissociating murine tissues at the maternal-fetal interface, and does not require the addition of other dissociating reagents or serum to terminate the dissociation reaction. Moreover, it is ready to use as supplied and, although the time of dissociation needs to be validated, it is more robust than the above-noted enzymes40,41.
The utilization of a combination of both types of tissue disaggregation improves the quality and amount of cells obtained; thus, several studies have implemented the combined use of mechanical and enzymatic dissociation with satisfactory results31,32,37. The protocol described herein was established and validated in our laboratory; it uses a combination of a gentle mechanical dissociation followed by a robust enzymatic disaggregation. This protocol allows the isolation and further study of the infiltrating leukocytes in murine tissues at the maternal-fetal interface (uterus, decidua, and placenta). The following protocol also maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as demonstrated by flow cytometric analysis. Finally, this protocol maintains the consistency of cell preparation for the analysis and comparison of different murine tissues composing the maternal-fetal interface.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Magentic Cell Separation</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30&#956;m pre separation filters</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multistand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL safe lock conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer</td>
			<td></td>
			<td></td>
			<td>(0.5% bovine serum albumin, 2mM EDTA and 1X PBS)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse extracellular antibodies</td>
			<td></td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td>(BSA)</td>
		</tr>
		<tr>
			<td>LIVE/DEAD viability dye</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation buffer solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td></td>
			<td></td>
			<td>(1% bovine serum albumin, 0.5% sodium azide, and 1X PBS ph 7.2)</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</td>
		</tr>
		<tr>
			<td>Incubator with shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from the murine tissues at the maternal-fetal interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

The dissection of murine tissues from the maternal-fetal interface is shown in Figure 1; this procedure includes opening the peritoneal cavity (Figure 1A,B), uterine horns (Figure 1C)&#160;including the implantation sites (Figure 1D), and the collection of the uterine tissues (Figure 1E), placenta (Figure 1F), and decidual tissues (Figure 1G) at 16.5 dpc. Figure 2 shows the morphology of...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface at JoVE.com

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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