This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH/DHHS. This work was also supported, in part, by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. &#160;We gratefully acknowledge Maureen McGerty (Wayne State University) for her critical readings of the manuscript.

This protocol is appropriate for leukocyte isolation from the decidua basalis and decidua parietalis in preparation for immunophenotyping by flow cytometry. Furthermore, the isolated cells can be used for cell sorting, cell culture, RNA isolation, and cytology. Before working with the samples mentioned in this protocol, human ethical approval must be obtained from the Local Research Ethics Committee and Institutional Review Boards. The collection and utilization of human samples for research purposes were approved by the Institutional Review Boards of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Department of Health and Human Services (DHHS, Bethesda, MD, USA) and Wayne State University (Detroit, MI, USA). Written informed consent was obtained from all pregnant women prior to the collection of tissue samples. 
NOTE: While working with animal blood, cells, or hazardous agents as mentioned in this protocol, it is essential that proper biosafety and laboratory safety actions be followed.
1. Dissection of the Human Decidual Tissues
NOTE: The basal plate is the base beneath and attached to the placenta and represents the maternal surface. The chorioamniotic membranes include the amnion and the chorion. The basal plate includes the decidua basalis and the chorion includes the decidua parietalis (Figure 1).
<ol>
	<li>Decidua Basalis
	<ol>
		<li>Dissect a piece of the basal plate from one cotyledon of the placenta (Figures 1A and 1B).</li>
		<li>Place it on a sterile cutting board with the placental villi facing upward. The side showing the placental villi is usually bloody red with hairy tissue in appearance. The basal plate is smooth and pale red in color.</li>
		<li>Use sharp, fine-point scissors and forceps to remove the villous tissue and blood vessels. Keep the tissue soaked in 1x PBS (Ca2+- and Mg2+- free) during the process (Figure 1C). Collect 2 to 3 of these pieces and rinse them thoroughly with 1x PBS to remove the blood (Figure 1D).</li>
	</ol>
	</li>
	<li>Decidua Parietalis
	<ol>
		<li>Dissect a piece of the chorioamniotic membranes (approximately 10 cm x 10 cm, Figure 1E). Place it on the board with the chorion facing upward. The chorionic side contains bloody clots and is usually light yellowish in color.</li>
		<li>Use fine-point forceps to remove as many of the blood clots as possible (Figure 1E). Rinse the membrane in sterile 1x PBS to clean the excess blood from the membrane until the 1x PBS runs clear.</li>
		<li>Use a disposable sterile cell scraper to gently scrape the decidual layer from the membrane. Apply sterile 1x PBS on the membrane while completing this process (Figure 1F). Collect the decidual tissues and place them in a 50 ml plastic tube with sterile 1x PBS (Figure 1G).</li>
	</ol>
	</li>
</ol>
2. Mechanical Disaggregation and Enzymatic Digestion
<ol>
	<li>Wash the tissue dissected from the decidua basalis (from Step 1.1.3) or the decidua parietalis (from Step 1.2.3) with sterile 1x PBS in a 50 ml plastic tube. Collect tissue pellets by centrifugation at 300 x g for 5 min at RT.</li>
	<li>Aspirate the supernatant located above the tissue pellet carefully without disturbing the pellet. 
	NOTE: At this point, the pellets are very loose because they contain red blood cells. If the volume of the pellet is about or less than 3 ml, re-suspend the pellet in 6 ml of cell detachment solution pre-warmed to 37 &#176;C. If the pellet is larger than 3 ml of volume, add more cell detachment solution (add about 2 times the volume of the tissue samples).</li>
	<li>Transfer the homogenized tissues (decidua basalis + cell detachment solution or decidua parietalis + cell detachment solution) to a C tube.</li>
	<li>Place the C tube in the automatic dissociator and run the corresponding program. 
	NOTE: The program for the decidua basalis runs for 17 sec with 668 total rounds per run. The program for the decidua parietalis runs for 37 sec with 235 total rounds per run. These programs have been customized to isolate leukocytes from the decidua basalis or the decidua parietalis with good yield and cell viability.</li>
	<li>Following the mechanical disaggregation of the tissues, digest the tissues with a commercially available cell detachment solution such as accutase; (a cocktail that contains proteolytic and collagenolytic enzymes) for 45 min at 37 &#176;C with gentle agitation.</li>
</ol>
3. Isolation of Leukocytes
<ol>
	<li>Following incubation, add 10 ml of 1x PBS to the digestion mixture and pass it through a 100 &#181;m cell strainer into a 50 ml plastic tube. Depending on the stickiness of the digestion mixture, one may need to use several cell strainers for one mixture.</li>
	<li>Fill the tube with 1x PBS and centrifuge at 300 x g for 5 min at RT. Remove the 1x PBS above the cell pellets carefully and re-suspend the pellets with 5 ml of ice-cold FACS buffer. 
	NOTE:To study the polymorphonuclear and mononuclear leukocytes together, go to Step 6.1; to study the mononuclear leukocytes, continue to Step 3.3.</li>
	<li>Add 5 ml of 20% density gradient media (1.077 + 0.001 g/ml) to a 15 ml plastic tube and slowly overlay the cell suspension on top of the density gradient media. While layering the sample, it is important not to mix the density gradient media with the cell suspension.</li>
	<li>Centrifuge with a swing-out rotor at 500 x g for 30 min at 4 &#730;C without the brake. It is very important to turn off the brake to avoid mixing the cells and losing the gradient. Leukocytes will be found in the interface between the density gradient media and the FACS buffer.</li>
	<li>Remove the upper layer that contains the FACS buffer and cell debris very carefully using a pipette. The band at the interface contains the decidual mononuclear cells and a portion of the polymorphonuclear leukocytes.</li>
	<li>Transfer the cells at the interface completely to a new 15 ml plastic tube. Add at least 3 times more volume of FACS buffer.</li>
	<li>Centrifuge at 300 x g for 5 min to pelletize the cells. Aspirate the supernatant and wash the cells with FACS buffer. Pelletize the cells with another centrifugation at 300 x g for 5 min at RT. 
	NOTE: To perform the cell culture, see Step 4. To perform cell sorting, see Step 5. To perform immunophenotyping, see Step 6.</li>
</ol>
4. Applications &#8211; Cell culture
<ol>
	<li>Re-suspend the cells from Step 3.7 in 1 ml of RPMI culture medium 1640. Count the viable cells using an automatic cell counter or a hemocytometer and a trypan blue solution 0.4%.</li>
	<li>Add up the amount of RPMI culture medium to make a cell concentration of 1 x 106 cells/ml. Cells are now ready for culture.</li>
</ol>
5. Applications &#8211; Isolation of Macrophages for Primary Cell Culture
<ol>
	<li>To isolate CD14+ cells, magnetically label the cells from Step 3.7 with CD14 microbeads (20 &#181;l beads per 107 cells), incubate for 15 min at 4 &#730;C, and separate CD14+ cells from other types of cells by applying a magnetic field. After 2 washes with MACS buffer and removal of the magnetic field, elute the magnetically retained CD14+ cells with MACS buffer.</li>
	<li>Following centrifugation, re-suspend the cell pellets in RPMI culture medium 1640. The cells are ready for plating (Figure 2).</li>
</ol>
6. Applications &#8211; Immunophenotyping
<ol>
	<li>To use the cells for immunophenotyping, re-suspend the cells from Step 3.7 in 1 ml of 1x PBS. Count the viable cells using an automatic cell counter or a hemocytometer and a trypan blue solution 0.4%.</li>
	<li>Label the cells with a fixable viability dye (Figure 3). Wash the cells twice with FACS buffer and re-suspend the cells in stain buffer. Incubate with an FcR blocker for 15 min at 4 &#176;C.</li>
	<li>Add extracellular antibodies conjugated to fluorochromes. For example, in this protocol, use anti-CD3, anti-CD19, anti-CD14, anti-CD15, and anti-CD56 antibodies (Table 1). Incubate for 30 min at 4 &#176;C in darkness.</li>
	<li>After 2 washes with 1x PBS, the cells will be analyzed in 500 &#181;l of stain buffer using a flow cytometer. A representation of the gating strategy used to analyze the leukocyte sub-populations found in the decidual tissues is shown in Figure 4.</li>
</ol>

The authors disclose no conflicts of interest.

Characterization of the functional and phenotypic properties of infiltrating leukocytes at the human maternal-fetal interface is essential to the understanding of the immune mechanisms that lead to&#160;pregnancy disorders. Several techniques have been described in order to isolate leukocytes from the human maternal-fetal interface throughout pregnancy 10,14,25,28,37,42,43. However, each of these techniques is distinct, uses different enzymes or enzyme combinations, requires different dissociation times, does ...

Pregnancy &#160;is characterized by three distinct immunological phases: 1) implantation and early placentation associated with a pro-inflammatory response (i.e., implantation resembles an &#8216;open wound&#8217;); 2) the second trimester and most of the third trimester of pregnancy when immune homeostasis is achieved through a predominantly anti-inflammatory state at the maternal-fetal interface; and 3) parturition, a pro-inflammatory state1-7. Immune cells play an important role in the regulation of the inflammatory response at the maternal-fetal interface where their abundance and localization change throughout pregnancy6-9.
In humans, the maternal-fetal interface represents an area of direct contact between maternal (decidua) and fetal (chorion or trophoblast) tissues. This interface includes: 1) the decidua parietalis that lines the uterine cavity not covered by the placenta and is in juxtaposition to the chorion laeve; and 2) the decidua basalis, located in the basal plate of the placenta where it is invaded by interstitial trophoblasts10 (Figure 1). The intimacy of these areas of contact creates conditions for fetal antigenic exposure to the maternal immune system11-13. Not surprisingly, leukocytes comprise up to 30-40% of the decidual cells8,9,14,15 in addition to typical stromal-type cells and glandular cells8,14,16. The role of leukocytes at the maternal-fetal interface encompasses multiple processes that include the limitation of trophoblast invasion17, remodeling of spiral arteries18,19, maintenance of maternal tolerance12,20, and initiation of labor21-26. Leukocytes of both the adaptive and innate limbs of the immune system, i.e., T cells, macrophages, neutrophils, B cells, dendritic cells, and NK cells, have been identified in the decidual tissues, and their proportions and activation status have been shown to vary spatially and temporally throughout gestation6-10,12,14,24,27-30. Perturbations in the leukocyte population and/or function are associated with&#160;spontaneous abortion31, preeclampsia32, intrauterine growth restriction32,33, and preterm labor7,24. Therefore, the study of the phenotypic characteristics and functionality of leukocytes at the human maternal-fetal interface will facilitate the elucidation of the immunological pathways dysregulated in pregnancy disorders.
One of the most powerful tools used to determine the phenotype and functional properties of leukocytes is flow cytometry, technology that allows the quantitative analysis of multiple parameters simultaneously34-36. To analyze leukocytes by flow cytometry, isolation of the leukocytes in a single-cell suspension is required. Therefore, a method to separate infiltrating leukocytes from the maternal-fetal interface is needed to study their phenotypic and functional properties.
Several methods have been described to isolate leukocytes from the human maternal-fetal interface10,14,25,27,37-39. While some apply mechanical disaggregation10,25,27,38, others use enzymatic digestion37,40 for tissue dissociation. Because mechanical disaggregation produces a lower yield and reduced viability41, and enzymatic dissociation can affect viability and cell surface marker retention42, the method described herein combines gentle mechanical dissociation with enzymatic pre-treatment to increase the yield of isolated leukocytes without compromising&#160;cell viability. A similar combination of methods has been demonstrated to be effective in the isolation of leukocytes from the decidual tissues at the maternal-fetal interface39. Therefore, the protocol described herein involves mechanical disaggregation with an automatic tissue dissociator that increases consistency while saving time and labor when compared to traditional mincing with opposing scalpels, razor blades, or surgical scissors10,28. The enzyme chosen for tissue dissociation was Accutase. Unlike commonly used collagenase43, dispase44, and trypsin45, Accutase (a cell detachment solution) combines both general proteolytic and collagenolytic activities that contribute to efficient yet gentle dissociation46,47. After dissociation, the leukocytes are enriched from the total population of the decidual cells by density gradient centrifugation. Various density gradient media have been previously utilized, the most common of which are Percoll (a suspension of colloidal silica particles)48 and Ficoll (a polymer of sucrose with a high synthetic molecular weight)49. The superior efficiency of isolation by the sucrose polymer has been previously shown50, and the protocol described herein further proves that this density gradient media produces a sufficiently high purity of mononuclear leukocytes.
Hence, the protocol described herein combines the mechanical tissue disaggregation with an automatic tissue dissociator, enzymatic digestion with a cell detachment solution, and leukocyte separation with a density gradient media (1.077 + 0.001 g/ml) to isolate leukocytes from human decidual tissues. This protocol has been proven to preserve cell surface antigens along with cell viability. The isolated leukocytes can be used for multiple applications that include immunophenotyping with flow cytometry and functional studies in vitro.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Dissection&lt;/strong&gt;</td></tr><tr><td>Sterile dissection tools: surgical scissors, forceps, and fine-tip tweezers&amp;nbsp;</td><td>Any vendor</td><td>20012-027</td><td></td></tr><tr><td>1X phosphate buffered saline (PBS)</td><td>Life Technologies</td><td></td><td>(1X PBS)</td></tr><tr><td>Large and small Petri dishes</td><td>Any vendor</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Dissociation&lt;/strong&gt;</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A11105-01</td><td>(cell detachment solution)</td></tr><tr><td>Sterile 2 ml safe-lock conical tubes</td><td>Any vendor</td><td></td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>Any vendor</td><td></td><td></td></tr><tr><td>100 &amp;micro;m cell strainers</td><td>FALCON/Corning</td><td>352360</td><td></td></tr><tr><td>5 ml round bottom polystyrene test tubes</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Transfer pipettes</td><td>Any vendor</td><td></td><td></td></tr><tr><td>C tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cell Culture&lt;/strong&gt;</td></tr><tr><td>RPMI culture medium 1640&amp;nbsp;</td><td>Life Technologies</td><td>22400-089</td><td>(1X) (10% FBS and 1% P/S)</td></tr><tr><td>Plastic chamber slides</td><td>Thermo Scientific</td><td>177437</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific Corporation</td><td>HEPA Class 100</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>ISOTEMP 110</td><td></td></tr><tr><td>Cell counter</td><td>Nexelcom</td><td>Cellometer Auto2000</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Olympus CKX41</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cell Separation&lt;/strong&gt;</td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Cell separator</td><td>Miltenyi Biotec</td><td>130-042-109</td><td></td></tr><tr><td>30 &amp;mu;m pre-separation filters</td><td>Miltenyi Biotec</td><td>130-041-40</td><td></td></tr><tr><td>Multistand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>15 ml safe-lock conical tubes</td><td>Any Vendor</td><td></td><td></td></tr><tr><td>MACS buffer&amp;nbsp;</td><td></td><td></td><td>(0.5% bovine serum albumin, 2 mM EDTA and 1X PBS)</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>FcR Blocking</td><td>Miltenyi Biotec</td><td>130-059-901</td><td>(Fc Block)</td></tr><tr><td>Anti-human cell surface antigen antibodies&amp;nbsp;</td><td>BD Biosciences</td><td></td><td>(Table 1)</td></tr><tr><td>Bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>LIVE/DEAD viability dye</td><td>BD Biosciences</td><td>564406</td><td></td></tr><tr><td>Lyse/Fix buffer&amp;nbsp;</td><td>BD Biosciences</td><td>346202</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td></td><td></td><td>(0.1% BSA, 0.05% Sodium Azide, and 1X PBS, pH=7.4)</td></tr><tr><td>Staining buffer&amp;nbsp;</td><td>BD Biosciences</td><td>554656</td><td></td></tr><tr><td>Trypan Blue solution 0.4%</td><td>Life Technologies</td><td>15250-011</td><td></td></tr><tr><td>Ficoll</td><td>GE Healthcare</td><td>17-1440-02</td><td>20% density gradient media (1.077 + 0.001 g/ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Additional Instruments&lt;/strong&gt;</td></tr><tr><td>Incubator with shaker</td><td>Thermo Scientific</td><td>MAXQ 4450</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>LSR-Fortessa</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>SpinChron DLX</td><td></td></tr><tr><td>Vacuum system</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Automatic tissue dissociator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>gentleMACS Dissociator</td><td></td></tr></tbody></table>

isolation of leukocytes from the human maternal-fetal interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

The dissection of human tissues at the maternal-fetal interface (decidua basalis and decidua parietalis) is shown in Figure 1. This procedure includes the dissection of the basal plate, which includes the decidua basalis (Figure 1A-D). The decidua basalis is obtained by removing the placental villi (fetal side) from the basal plate (Figure 1C). The decidua parietalis is collected by gently scraping the chorionic membrane (Figure 1E-<stro...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Human Maternal-fetal Interface at JoVE.com

Isolation of Leukocytes from the Human Maternal-fetal Interface

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

isolation-of-leukocytes-from-the-human-maternal-fetal-interface

Research

JoVE Journal

Immunology and Infection

15.9K Views.  NICHD/NIH/DHHS. Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis. 

Video: Isolation of Leukocytes from the Human Maternal-fetal Interface

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Developmental Biology

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Biology

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Bioengineering

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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.

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 ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Medicine

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...