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

Summary

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.

Abstract

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.

Introduction

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 rejected^4,5. This enigma is not fully understood.

The most recent hypothesis states that maternal-fetal tolerance is created during coitus and fecundation^6,7 and maintained to sustain a full-term pregnancy^8-10. A breakdown ....

Protocol

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 small surgical scissors, forceps, and fine-tip tweezers. Include small Petri dishes labeled with the appropriate tissue names, filled with 1x phosphat....

Results

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 isol.......

Discussion

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 pregnancy^{31,38,39,43-46}. However, each technique is different, uses different enzymes or enzyme combinations, requires.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Magentic Cell Separation
MS Columns	Militenyi Biotec	130-042-201
Cell Separator	Militenyi Biotec	130-042-102
30 μm pre-separation filters	Militenyi Biotec	130-041-407
Multistand	Militenyi Biotec	130-042-303
15 ml conical tubes	Thermo Fisher	339650
MACS Buffer	Militenyi Biotec	Laboratory preparation	(0.5% bovine serum albumin, 2mM EDTA and 1x PBS, pH 7.2)
Reagents
Accutase enzymatic solution	Life Technologies	A11105-01
Anti-mouse CD16/CD32	BD Biosciences	533142
Anti-mouse extracellular antibodies	BD Bioscences/eBiosciences		Table 1
Sodium azide	Fisher	S227-500
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906-100G
LIVE/DEAD viability dye	Life Technologies	L23105
Fixation buffer solution	BD Biosciences	558049
FACS Buffer	BD Biosciences	Laboratory preparation	0.1% BSA, 0.05% sodium azide, 1x PBS, pH 7.4
Trypan blue solution 0.4%	BIO-RAD	145-0013
Fetal bovine serum (FBS)	Life Technologies	16000-044
Additional Instruments
Incubator with shaker	Thermo Scientific	MaxQ 4450
Flow cytometer	BD LSRFortessa
Centrifuge	Thermo Scientific	Legend LXTR
Vacuum system			laboratory constructed
Incubator	Thermo Scientific	Incubator 3307
Water bath	Precision	51221035
Cell counter	BIO-RAD	TC20 Automated Cell Counter
Optical and fluorescence microscopes	Zeiss and Olympus	Zeiss LSM780 and Olympus CKX41