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Immunology and Infection

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

Published: May 21st, 2015

DOI:

10.3791/52866

1Department of Obstetrics & Gynecology, Wayne State University School of Medicine, 2School of Paediatrics and Reproductive Health, Research Centre for Reproductive Health, the Robinson Research Institute, The University of Adelaide, 3Department of Immunology & Microbiology, Wayne State University School of Medicine, 4Perinatology Research Branch, NICHD/NIH/DHHS

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 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 (>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.

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 ....

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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

  1. Prepare a sterile workstation and obtain sterile tools for tissue collection. These tools will include large and .......

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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) 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.......

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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.......

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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.

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NameCompanyCatalog NumberComments
Magentic Cell Separation
MS Columns
Cell Separator
30μm pre separation filters
Multistand
15mL safe lock conical tubes
MACS Buffer(0.5% bovine serum albumin, 2mM EDTA and 1X PBS)
Reagents
Anti-mouse CD16/CD32
Anti-mouse extracellular antibodies(Table 1)
Sodium azide
Bovine serum albumin(BSA)
LIVE/DEAD viability dye
Fixation buffer solution
FACS Buffer(1% bovine serum albumin, 0.5% sodium azide, and 1X PBS ph 7.2)
Trypan Blue Solution 0.4%
Fetal bovine serum
Additional Instruments
Incubator with shaker
Flow cytometer
Centrifuge
Vacuum system
Incubator
Water bath
Cell counter
Microscope

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