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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Sterile dissection tools: surgical scissors, forceps, and fine-tip tweezers&#160;</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></td>
			<td></td>
			<td></td>
			<td>Dissociation</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 &#181;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></td>
			<td></td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>RPMI culture medium 1640&#160;</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></td>
			<td></td>
			<td></td>
			<td>Cell Separation</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&#956;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>15mL safe-lock conical tubes</td>
			<td>Any Vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS buffer&#160;</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>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&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Bovine serum albumin&#160;</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&#160;</td>
			<td>BD Biosciences</td>
			<td>346202</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td></td>
			<td></td>
			<td>(1% BSA, 0.5% Sodium Azide, and 1X PBS)</td>
		</tr>
		<tr>
			<td>Staining buffer&#160;</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</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</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&#160;</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&#160;</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 Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

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 ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...