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

Before working with the samples mentioned in this protocol, animal ethical approval must be given by the Local Research Ethics Committee and the Institutional Review Boards. When working with animal blood, cells, or hazardous agents as mentioned in this protocol, the proper biosafety and laboratory safety actions must be followed.
1. Mouse Handling and Tissue Collection
<ol>
	<li>Prepare a sterile workstation and obtain sterile tools for tissue collection. These tools will include large and small surgical scissors, forceps, and fine-tip tweezers. Include small Petri dishes labeled with the appropriate tissue names, filled with 1x phosphate-buffered saline (PBS) solution (3 - 5 ml) equilibrated to RT (20 - 22 &#176;C). Also include a large Petri dish, unlabeled, filled with 1x PBS solution (10 - 15 ml).</li>
	<li>Euthanize a pregnant mouse (10.5 to 19.0 days post-coitum (dpc) or prior to delivery) using carbon dioxide (CO2). To ensure that the mouse is euthanized, use the cervical dislocation technique. 
	Note: Prior to 10.5 dpc, it is difficult to manually separate the decidual tissues from the uterine tissues. If the isolation of decidual leukocytes is not required, leukocytes from the uterine tissues of non-pregnant mice or those from mice at &#60;10.5 dpc can also be performed by following this protocol.</li>
	<li>Using a pair of sterile surgical scissors, completely remove the lower abdominal portion of the skin and muscular tissue. With forceps, move aside all other organs until the uterus and ovaries are visible. With the uterus still attached, use the forceps to move it outside of the peritoneal cavity (Figure 1A).</li>
	<li>Locate the ovaries distal to the oviduct and uterotubal junction of each uterine horn. Using the small surgical scissors, make an incision at the uterotubal junction and separate the uterine horns from the mesentery containing the uterine vessels; then, excise at the cervix to detach the uterine horns from the peritoneal cavity (Figure 1B).</li>
	<li>With small surgical scissors, make an incision between the cervix and the lower segment of the uterine horns. Leave attached a portion of the cervix during this process (Figure 1C) to avoid opening the uterine horn.</li>
	<li>Immerse the uterine horns (including the cervix) in a large Petri dish filled with 1x PBS solution (10 - 15 ml) to hydrate the implantation sites.</li>
	<li>While still immersed in 1x PBS solution, trim any fat from the uterine horns with the small surgical scissors. Keep the tissues immersed in 1x PBS solution throughout the dissection.</li>
	<li>Holding the uterine horns in place with the forceps, remove one of the implantation sites from the uterus with a transverse cut through the inter-implantation region, using the small surgical scissors (Figure 1D). The implantation site contains the fetus, surrounded by the chorioallantoic membrane, and the uterine, decidual, and placental tissues.</li>
	<li>Using the forceps, hold the site in place and make a small incision in the uterine wall adjacent to the placental and decidual tissues (Figure 1E). Trim around the perimeter of the placenta/decidua until these become separated from both the uterine wall and the chorioallantoic membrane that includes the fetus (Figure 1F).</li>
	<li>Remove the placenta and attached decidual tissues and place in 1x PBS solution (3 - 5&#160; ml; see step 1.12).</li>
	<li>Place the fetus surrounded by the chorioallantoic membrane attached to the uterine tissues in a Petri dish filled with 1x PBS solution (3 - 5&#160; ml). The hydration of these tissues will facilitate the separation of the chorioallantoic membrane from the uterine wall (see step 1.14).</li>
	<li>Using a pair of fine-tip tweezers, gently peel the decidual tissue (white-grey layer) from the placental surface. Perform this step carefully to maintain the integrity of the placental and decidual tissues (Figure 1G).</li>
	<li>Place the placenta and decidual tissues in two separately labeled Petri dishes filled with 1x PBS solution (3 - 5&#160; ml each).</li>
	<li>Gently remove the uterine wall from the chorioallantoic membrane with fine-tip tweezers. These are the uterine tissues.</li>
	<li>Place the uterine tissues in a Petri dish filled with 1x PBS solution (3 - 5&#160; ml).</li>
	<li>Repeat steps 1.8 through 1.15 until all implantation sites have been processed.</li>
	<li>Collect several uterine, decidual, and placental tissues to improve the yield of cells during enzymatic digestion. The amount of tissues depends on the litter size; however, leukocyte isolation requires &#62;2 pieces of tissue. 
	Note: For more detailed information regarding the dissection of the implantation sites on different gestational days, view the pictures found in Chapter Two of The Guide to Investigation of Mouse Pregnancy42.</li>
</ol>
2. Uterine, Decidual, and Placental Tissue Dissociation
<ol>
	<li>Label several sterile 2 ml conical tubes with the appropriate tissue name, and place a vial with 1,000 &#181;l&#160;of enzymatic solution on ice.</li>
	<li>From the Petri dish filled with 1x PBS solution, transfer two pieces (100 - 150 mg) of the uterine, decidual, or placental tissues to a labeled 2 ml conical tube.</li>
	<li>Add 500 &#181;l of cold enzymatic solution into each 2 ml conical tube.</li>
	<li>With fine sterile scissors, begin to dissociate the tissue immersed in the enzymatic solution until it is finely minced. Over time, the suspension will develop a milky appearance. This step should not exceed more than 2 min to prevent over-manipulation of the sample.</li>
	<li>Once the tissue has been dissociated, add an extra 500 &#181;l of cold enzymatic solution to the 2 ml conical tube.</li>
	<li>Place the 2 ml conical tube on ice.</li>
	<li>Repeat steps 2.2 through 2.6 with each individual tissue until all tissues have been processed.</li>
	<li>Transfer all 2 ml conical tubes with homogenized tissue (minced tissue + enzymatic solution) samples from the ice into an incubator at 37&#160;&#186;C and perform a gentle orbital agitation (80 rpm). Incubate for 30 - 35 min. Make sure the temperature of the incubator is stabilized before starting the incubation time.</li>
	<li>Following incubation, remove the 2 ml conical tubes with dissociated tissues from the incubator and place them on ice to prevent any further digestion.</li>
	<li>Label sterile 50 ml conical tubes according to tissue type. Remove the lids and replace them with a sterile cell strainer (100 &#181;m pore size).</li>
	<li>Pour the dissociated tissues from the 2 ml conical tubes through the cell strainer using ~20 ml of 1x PBS solution. The fragments of tissue will remain in the cell strainer and the cell suspension will pass through it.</li>
	<li>Wash the dissociated tissues in the cell strainer 2 - 3 times with 20 ml of 1x PBS solution using a transfer pipette.</li>
	<li>Rinse the empty 2 ml conical tubes with 1x PBS solution (1 ml) and pour the contents through the cell strainers.</li>
	<li>Remove the cell strainers from the 50 ml conical tubes and replace the lids. Centrifuge the tubes at 1,250 x g for 10 min at 4 &#186;C.</li>
	<li>Carefully, aspirate the supernatant without disturbing the cell pellet. The cell pellet contains the isolated leukocytes and stromal cells.</li>
	<li>Re-suspend the cell pellet in 1 ml of RPMI culture medium without fetal bovine serum (FBS). Mix gently. Do not use vortex.</li>
	<li>Add 500 &#181;l of neat FBS to a 5 ml polystyrene plastic tube and slowly overlay the cell suspension.</li>
	<li>Centrifuge for 10 min at 1,100 x g without the brake at RT to pellet viable cells while cellular debris is retained at the PBS/FBS interface.</li>
	<li>Aspirate the supernatant without touching the cell pellet. The cell pellet contains mostly viable cells.</li>
	<li>Optionally: To concentrate the mononuclear cells, use a 20% saturated polysaccharide solution, as described below.
	<ol>
		<li>Resuspend the cell pellet in 1,000 &#181;l of RPMI culture medium without FBS.</li>
		<li>Add 1 ml of 20% saturated polysaccharide solution to a 5 ml polystyrene plastic tube and slowly overlay the cell suspension on top of this solution, according to the manufacturer&#8217;s instructions. While layering the sample, avoid mixing the 20% saturated polysaccharide solution with the cell suspension.</li>
		<li>Centrifuge with a swing-out rotor at 500 x g for 30 min at RT without brake. It is very important to turn off the brake to avoid mixing the cells and losing the gradient. Mononuclear cells will be found in the interface between the 20% saturated polysaccharide solution and the 1x PBS solution.</li>
		<li>Aspirate and discard the supernatant. The cell pellet contains mostly mononuclear cells. 
		Note: To perform viability staining and intracellular staining, see step 3. To perform immunophenotyping, see step 4. To perform viability staining and extracellular staining only, see step 5. To perform a cell culture of isolated leukocytes, see step 6. To perform magnetic cell sorting, see step 7.</li>
	</ol>
	</li>
</ol>
3. Viability Staining for Fixable Cells
<ol>
	<li>Resuspend the cell pellet in 1,100 &#181;l of 1x PBS solution. Mix gently using a micro-pipette.</li>
	<li>Transfer 100 &#181;l of the cell suspension into a 5 ml polystyrene plastic tube. Add 400 &#181;l of 1x PBS solution to the 5 ml polystyrene tube. This tube is a control for tissue auto-fluorescence. Store at 4 &#186;C until step 3.5.</li>
	<li>Transfer the remaining 1,000 &#181;l of the cell suspension to a new 5 ml polystyrene plastic tube. Add 1 &#181;l of the viability dye and gently homogenize. Incubate for 30 min in darkness at 4 &#186;C.</li>
	<li>Following incubation, add 1,000 &#181;l of 1x PBS solution. Mix gently by hand.</li>
	<li>Centrifuge both 5 ml polystyrene plastic tubes (steps 3.2 and 3.3) at 1250 x g for 10 min at 4 &#186;C. Aspirate and discard the supernatant. 
	Note: The cell pellet (step 3.3) will be used in step 4. The cell pellet (step 3.2) will serve as a control for tissue auto-fluorescence.</li>
</ol>
4. Immunophenotyping
<ol>
	<li>Resuspend the cell pellet in 50 &#181;l of anti-mouse CD16/CD32 (diluted 1:100 in FACS buffer [0.1% BSA, 0.05% sodium azide, 1x PBS solution, ph 7.4]). Mix the cell suspension gently. Do not use the vortex.</li>
	<li>Incubate the cell suspension in the 5 ml polystyrene tube in darkness at 4 &#176;C for 10 min.</li>
	<li>Following incubation, add 50 &#181;l of each anti-leukocyte monoclonal antibody reacting with extracellular cell surface markers or isotype-matched control antibody (antibody dilutions must be validated). For example, to analyze leukocyte sub-populations, use anti-mouse antibodies reacting with the following leukocyte-cell surface markers: CD45, Ly6G, F4/80, CD11c, CD49b, B220, CD3, CD4, and CD8 (Table 1).</li>
	<li>Once antibodies against extracellular markers have been added to the 5 ml polystyrene plastic tube, mix gently. Incubate in darkness for 30 min at 4 &#176;C.</li>
	<li>During this incubation, prepare and pre-warm the fixation buffer solution (diluted with deionized water, 1:5) to 37 &#186;C, if required. Keep the buffer at 37 &#186;C in darkness until its use is necessary.</li>
	<li>After 30 min incubation, add 500 &#181;l of FACS buffer in the 5 ml polystyrene plastic tube and gently mix. This step is needed to wash the cells and remove any excess of unbound antibody.</li>
	<li>Centrifuge the samples at 1,250 x g at 4 &#176;C for 10 min. Aspirate and discard the supernatant. 
	Note: If intracellular staining is not required, see step 5. If intracellular staining is required, perform permeabilization and intracellular staining here, and then proceed with the fixation (step 4.8).</li>
	<li>Add 500 &#181;l of the pre-warmed fixation buffer solution to the cell pellet and incubate in darkness for 10 min at 37 &#186;C.</li>
	<li>Dispense 500 &#181;l of FACS buffer into the 5 ml polystyrene plastic tube. Mix gently.</li>
	<li>Centrifuge the samples at 1,250 x g at 4 &#176;C for 10 min. Aspirate and discard the supernatant.</li>
	<li>Add 500 &#181;l of FACS buffer to each sample. These samples can now be analyzed by flow cytometry. Acquire the samples immediately for best results.</li>
</ol>
5. Viability Staining for Unfixable Cells
<ol>
	<li>After extracellular immunophenotyping, resuspend the cell pellet in 500 &#181;l of FACS buffer.</li>
	<li>Add 1 &#181;l of 4&#8217;,6-diamidino-2-phenylindole dihydrochloride (DAPI, 200 &#181;g/ml) just before acquiring the sample.</li>
	<li>Visualize the samples within 1 - 2 min after adding DAPI using a flow cytometer.</li>
</ol>
6. Cell Culture
<ol>
	<li>Prepare a workstation under a sterile fume hood. Pre-warm the RPMI culture medium to 37 &#176;C using a water bath.</li>
	<li>Resuspend the cell pellet in 1000 &#181;l of FACS buffer for cell counting.</li>
	<li>Count the number of viable cells using an automatic cell counter or hemocytometer and 0.4% trypan blue solution, following the manufacturer&#39;s instructions. Record the total live cell count.</li>
	<li>Centrifuge the cells at 1,250 x g at 4 &#176;C for 10 min to remove the FACS buffer.</li>
	<li>Resuspend the cell pellet in 500 &#181;l of pre-warmed RPMI culture medium by gently pipetting 2 to 3 times.</li>
	<li>Plate the desired number of cells in a sterile 24-well plate and incubate at 37 &#176;C. The incubation time will be determined by the research question.</li>
</ol>
7. Magnetic Cell Sorting
<ol>
	<li>Set up a clean workstation with the following materials: MS columns, magnetic cell separator, 15 ml conical tubes, 30 &#181;m pre-separation filters, and a multi-stand.</li>
	<li>Resuspend the cell pellet in 500 &#181;l of MACS buffer (0.5% BSA, 2mM EDTA, 1x PBS solution, pH 7.2).</li>
	<li>Using an automatic cell counter or hemocytometer and 0.4% trypan blue solution, count and record the total live cell count.</li>
	<li>Centrifuge the cells at 1,250 x g at 4 &#176;C for 10 min. Aspirate and discard the supernatant. Add another 500 &#181;l of MACS buffer.</li>
	<li>Proceed to perform the magnetic separation following the manufacturer&#8217;s recommendations.</li>
	<li>Determine purity by flow cytometry or microscopy.</li>
</ol>

The authors disclose no conflicts of interest.

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 different dissociation times, does not specify quantities of tissue, and, most importantly, does not always specify the viability of the isolated cells. The protocol described herein allows the isolation of infiltrating leukocytes from the murine tissues at the maternal-fetal interface with high viability, and provides detailed information about the commercial reagents, buffer preparation, tissue quantities, and incubation times validated in the laboratory.
One of the most critical steps of the leukocyte isolation process is the tissue dissociation; this step involves mechanical homogenization and/or enzymatic reactions that can alter the integrity of extracellular proteins used in phenotypic characterization47. The protocol described herein offers a novel approach that combines the use of gentle mechanical and enzymatic tissue dissociation techniques to preserve the integrity of extracellular markers in the leukocytes isolated from the placenta and the decidual and uterine tissues of mice.
Single enzymes and combinations of different enzymes have been used to isolate infiltrating leukocytes from the murine tissues at the maternal-fetal interface31,32,39,44. In many cases, these enzymes that are prepared to specific concentrations by hand in the laboratory may be subject to human error. Here, instead, a ready-to-use purified collagenase/neutral protease cocktail, Accutase, has been implemented in the laboratory; as a commercially available enzyme preparation, it has been shown to provide reliable results in cell culture48. This enzymatic solution is known to effectively detach macrophages from the culture plates without scraping and, most importantly, without losing surface antigens48. This prepared enzyme has also been used to process the digestion of human and animal nervous system tissues, resulting in viable isolated cells that remain sustainable for long periods, which allows their subsequent culture47. Moreover, this enzymatic solution preserves CD24 antigenicity in isolated cells from central nervous system tissues47. When compared to Liberase-1, another cocktail of collagenase and neutral protease, neither Accutase nor Liberase-1 generate free DNA aggregates; however, Accutase is gentler than Liberase-1 during tissue dissociation47. The collagenase/neutral protease cocktail used in this protocol has also demonstrated superiority to trypsin in the preservation of CD44, a cancer stem cell surface marker49. This laboratory&#8217;s studies have consistently noted that this enzymatic solution preserves mouse leukocyte surface antigens. Indeed, informative differences have been found in the expression of extracellular markers in macrophages (CD11b+F4/80+ cells), neutrophils (CD11b+Ly6G+ cells), NKT cells (CD3+CD49b+ cells), T cells (CD3+ cells) and B cells (B220+CD19+ cells), including CD4, CD8, CD69, CD25, CD40L, PD1, CD44, CD62L, and CTLA4, and in cytokine release. Therefore, the method described herein is optimal for immunophenotyping of infiltrating leukocytes at the maternal-fetal interface in mice, as shown in the representative results.
One important advantage of this method is the simultaneous determination of several extracellular and intracellular antigens within the viability gate. The dye most used to determine cell viability is propidium iodide (PI); however, its use is limited since it can be used only in combination with FITC. This is because PI cannot be adequately distinguished from most other fluorochromes excited by blue and red lasers50. A solution is to use DAPI50 or any other dye that is excited by UV lasers but not by blue and red lasers commonly used for immunophenotyping. This protocol allows immunophenotyping of viable cells as it includes the use of DAPI50 for unfixable cells or the use of viability dye for fixable cells.
A second important advantage of the protocol described herein is that it allows the isolation of leukocytes with a high yield of viable cells. The representative data shows that 70% to 89% of the isolated cells are viable. This is of great importance as this protocol has allowed the study of the functional properties of the isolated cells from murine tissues at the maternal-fetal interface. For example, cultures of the decidual and uterine macrophages and the study of cytokine release under stimulation have been performed using this method.
To achieve successful results, the application of the described protocol is important when considering the following factors: 1) tissue collection must be performed within 5 to 10 min&#160;after opening the peritoneal cavity, and these tissues must be placed on ice to preserve the viability of the isolated cells; 2) mechanical tissue homogenization must be performed using fine-tip scissors and cannot exceed 2 min&#160;since longer periods have been demonstrated to reduce the yield of viable cells; 3) the duration of incubation with the enzymatic solution must be less than 1 hr&#160;since its activity diminishes after this period of time; 4) the temperature of incubation with the enzymatic solution must be maintained at 37 &#176;C to obtain the optimal activity of this cocktail of enzymes; 5) cell pellet manipulation must be done gently with micropipettes because use of the vortex can damage the integrity of the cells (isolated cells are differentiated and abrupt manipulation can easily reduce their viability); 6) buffer and centrifuge temperatures must be kept at the same temperature as the cell suspension; 7) isolated cells must be processed for immunophenotyping or used immediately as their viability reduces rapidly; and 8) when performing immunophenotyping, samples must be acquired using a flow cytometer immediately for best results.
A limitation of this protocol is the cost of the enzymatic solution, which is more expensive than other enzymes with similar functions, e.g., trypsin, dispase II, and collagenase. However, the advantages that this enzymatic solution displays over and above the described enzymes are superior.
Besides immunophenotyping and cell culture and sorting, the future applications of this protocol are numerous and varied. For example, it is now possible to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (e.g., phagocytosis, cytotoxicity, T-cell proliferation and plasticity assays, etc.), and production of reactive oxygen species&#160;in the leukocytes isolated from murine tissues at the maternal fetal interface. Indeed, description of new and rare leukocytes in murine tissues at the maternal-fetal interface can be also performed.

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

<table><tbody><tr><td colspan="4">&lt;strong&gt;Magentic Cell Separation&lt;/strong&gt;</td></tr><tr><td>MS Columns</td><td>Militenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Cell Separator</td><td>Militenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>30 &amp;mu;m pre-separation filters</td><td>Militenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Multistand</td><td>Militenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Thermo Fisher</td><td>339650</td><td></td></tr><tr><td>MACS Buffer</td><td>Militenyi Biotec</td><td>Laboratory preparation</td><td>(0.5% bovine serum albumin, 2mM EDTA and 1x PBS, pH 7.2)</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Accutase enzymatic solution</td><td>Life Technologies</td><td>A11105-01</td><td></td></tr><tr><td>Anti-mouse CD16/CD32</td><td>BD Biosciences</td><td>533142</td><td></td></tr><tr><td>Anti-mouse extracellular antibodies</td><td>BD Bioscences/eBiosciences</td><td></td><td>&lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>Sodium azide</td><td>Fisher</td><td>S227-500</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906-100G</td><td></td></tr><tr><td>LIVE/DEAD viability dye</td><td>Life Technologies</td><td>L23105</td><td></td></tr><tr><td>Fixation buffer solution</td><td>BD Biosciences</td><td>558049</td><td></td></tr><tr><td>FACS Buffer</td><td>BD Biosciences</td><td>Laboratory preparation</td><td>0.1% BSA, 0.05% sodium azide, 1x PBS, pH 7.4</td></tr><tr><td>Trypan blue solution 0.4%</td><td>BIO-RAD</td><td>145-0013</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></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 LSRFortessa</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>Legend LXTR</td><td></td></tr><tr><td>Vacuum system</td><td></td><td></td><td>laboratory constructed</td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>Incubator 3307</td><td></td></tr><tr><td>Water bath</td><td>Precision</td><td>51221035</td><td></td></tr><tr><td>Cell counter</td><td>BIO-RAD</td><td>TC20 Automated Cell Counter</td><td></td></tr><tr><td>Optical and fluorescence microscopes</td><td>Zeiss and Olympus</td><td>Zeiss LSM780 and Olympus CKX41</td><td></td></tr></tbody></table>

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

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

The dissection of murine tissues from the maternal-fetal interface is shown in Figure 1; this procedure includes opening the peritoneal cavity (Figure 1A,B), uterine horns (Figure 1C)&#160;including the implantation sites (Figure 1D), and the collection of the uterine tissues (Figure 1E), placenta (Figure 1F), and decidual tissues (Figure 1G) at 16.5 dpc. Figure 2 shows the morphology of isolated macrophages (F4/80+) collected from the decidual and uterine tissues at 16.5 dpc using magnetic cell sorting. Isolated macrophages maintain the ability to release cytokines (data not shown). The yield of viable cells isolated from the decidua, uterus, and placenta is shown in Figure 3, and cell viability is greater than 70% in all tissues. Figure 4 shows the gating strategy for analyzing polymorphonuclear and mononuclear leukocytes within the singlets and viability gates, including T cells (CD45+CD3+), neutrophils (CD45+Ly6G+), macrophages (CD45+F4/80+), dendritic cells (CD45+CD11c+), and NK cells (CD45+CD49b+) at 16.5 dpc. A high proportion of macrophages co-express CD11c. Figure 5 shows neutrophils, macrophages, and T cells in the decidual, uterine, and placental tissues at 16.5 dpc. Figure 6 shows the gating strategy for analyzing lymphocytes within the singlets and viability gates, including T cells (CD45+CD3+) and B cells (CD45+B220+) at 16.5 dpc. T cells include CD4+ and CD8+ T cells. Murine tissues at the maternal-fetal interface also include CD3+CD4-CD8- (gamma-delta&#160;T cells) in high proportions. Figure 7 shows T cells and B cells in the decidual, uterine, and placental tissues at 16.5 dpc.
<img alt="Figure 1" src="/files/ftp_upload/52866/52866fig1highres.jpg" width="700" /> 
Figure 1. Tissue dissection. (A) Uterine horns at 16.5 dpc in a B6 mouse. The uterine horns are attached to the mesentery and draining vessels. (B) Uterine horns dissected from the mesentery and still attached to the cervix. (C) Uterine horns including the implantation sites and cervix. (D) Dissection of an implantation site. (E) Dissection of the uterine tissues from the implantation site. (F) Separation of the placenta and decidua from the implantation site. (G) Detachment of the decidua (white-gray layer) from the placenta. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52866/52866fig2highres.jpg" width="500" /> 
Figure 2. Macrophages isolated from decidual and uterine tissues. Macrophages (F4/80+ cells) isolated from the decidual and uterine tissues at 16.5 dpc using magnetic cell sorting. 20X magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52866/52866fig3highres.jpg" width="600" /> 
Figure 3. Viability of isolated cells. Viable cells (DAPI- cells represented with arrows) isolated from decidual, uterine, and placental tissues. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52866/52866fig4highres.jpg" width="700" /> 
Figure 4. Gating strategy for polymorphonuclear and mononuclear leukocytes. Total leukocyte population was gated within the singlets and viability gates. T cells (CD45+CD3+), macrophages (CD45+F4/80+), neutrophils (CD45+Ly6G+), dendritic cells (CD45+CD11c+), and NK cells (CD45+CD49b+) were gated within the total-leukocyte gate (CD45+). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52866/52866fig5highres.jpg" width="600" /> 
Figure 5. Neutrophils, macrophages, and T cells in murine tissues at the maternal-fetal interface. Neutrophils (CD45+Ly6G+), macrophages (CD45+F4/80+), and T cells (CD45+CD3+) were gated within the viability and total-leukocyte (CD45+) gates in isolated decidual, uterine, and placental cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52866/52866fig6highres.jpg" width="600" /> 
Figure 6. Gating strategy for lymphocytes. Mononuclear cells were gated within the singlets and viability gates. T cells (CD3+) and B cells (B220+) were gated within the viability gate. CD4+ and CD8+ T cells were gated within the T-cell gate (CD3+). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52866/52866fig7highres.jpg" width="600" /> 
Figure 7. Lymphocyte sub-populations in murine tissues at the maternal-fetal interface. T cells (CD3+) and B cells (B220+) were gated within the viability gate in isolated decidual, uterine, and placental cells. CD4+ and CD8+ T cells were gated within the T-cell gate (CD3+). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52866/52866fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell marker</td>
			<td>Fluorochrome</td>
			<td>Clone</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>LIVE/DEAD</td>
			<td>DAPI</td>
			<td>-</td>
			<td>Life Technologies</td>
			<td>L23105</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>V450</td>
			<td>30-F11</td>
			<td>BD Biosciences</td>
			<td>560501</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>FITC</td>
			<td>145-2C11</td>
			<td>BD Biosciences</td>
			<td>553062</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>APC</td>
			<td>RM4-5</td>
			<td>BD Biosciences</td>
			<td>553051</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>PE-CF594</td>
			<td>53-6.7</td>
			<td>BD Biosciences</td>
			<td>562283</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>APC-Cy7</td>
			<td>RA3-6B2</td>
			<td>BD Biosciences</td>
			<td>552094</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>PE</td>
			<td>BM8</td>
			<td>eBiosciences</td>
			<td>12-4801-82</td>
		</tr>
		<tr>
			<td>Ly6G</td>
			<td>APC-Cy7</td>
			<td>1A8</td>
			<td>BD Biosciences</td>
			<td>560600</td>
		</tr>
		<tr>
			<td>CD49b</td>
			<td>APC</td>
			<td>DX5</td>
			<td>BD Biosciences</td>
			<td>560628</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>PE-Cy7</td>
			<td>HL3</td>
			<td>BD Biosciences</td>
			<td>558079</td>
		</tr>
	</tbody>
</table>
Table 1. List of antibodies utilized for leukocyte subset immunophenotyping

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

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

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

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

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.2K Views.  Wayne State University School of Medicine. 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. 

Video: Isolation of Leukocytes from the Murine Tissues at the 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 of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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.

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 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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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 ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Isolation of Lymphocytes from a Mouse Peyer&#39;s Patch

Begin by placing a mouse in the supine position, and sterilizing the abdomen with 70% ethanol. Make a midline abdominal incision through the skin and peritoneum from the pubis to the rib cage to open the peritoneal cavity and locate the cecum and the ileocecal junction.
Make as distal as possible cut, at the junction to separate the small intestine from the cecum, and cut the mesentery with scissors to carefully remove the entire small intestine up to the pyloric sphincter.
Snip the junction between the pylorus and the duodenum to completely detach the small intestine from the abdominal cavity, and place the isolated small intestine into one well of a six-well plate containing cold RPMI medium, supplemented with 10% Fetal Bovine Serum or FBS on ice. Then, gently agitate the tissues manually until all of the segments are submerged in the cold medium.
When all of the intestines have been harvested, use forceps to grasp the mesenteric fat of one edge of the intestine and place the sample &#8212; mesenteric side down &#8212; on a paper towel. Moisten the entire intestinal segment with RPMI plus 10% FBS to avoid tissue dehydration and stickiness, and locate the Peyer&#39;s patches, which appear as white multilobulated aggregates with a cauliflower-like shape on the anti-mesenteric side of the intestinal wall.
To harvest the Peyer&#39;s patches, use curved surgical scissors with the curve facing up to gently excise each patch from its distal and proximal borders, taking care to exclude the surrounding intestinal tissue, and place the Peyer&#39;s patches into individual wells of a 12-well plate containing ice-cold RPMI plus 10% FBS, on ice, as they are collected.
When all of the patches have been acquired, use scissors to cut the tip of a 1-milliliter micropipette tip so the diameter is large enough to aspirate individual Peyer&#39;s patches, and transfer the Peyer&#39;s patches into 150-milliliter conical tube per mouse, containing 25-milliliters of 37 degrees Celsius RPMI plus 10% FBS.
Then, place the tubes in an orbital shaker at 37 degrees Celsius with continuous agitation at 125 to 150 RPM for 10 minutes. At the end of the agitation, transfer the Peyer&#39;s patches onto 140-micrometer cell strainer per mouse, and use the rubber end of 110-milliliter syringe plunger per animal to gently disrupt the Peyer&#39;s patches through the mesh into individual 50-milliliter conical tubes.
Rinse the strainers with 15 to 20 milliliters of cold RPMI plus 10% FBS, and collect the cells by centrifugation. After carefully discarding the supernatant, re-resuspend the cells at an approximately 1 x 107 cells per milliliter of RPMI plus 10% FBS concentration for counting. Then, transfer 2 to 2.5 x 106 cells per 200 micro-liters of medium, to each well of a 96-well round bottom plate, and pellet the cells by centrifugation.

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

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