Study supported by NIH grants AI102838 and AI117739 (CRW). We thank Richard Rossoll for technical assistance. Flow cytometric analysis was carried out in DartLab, the Shared Resource at Dartmouthsupported by (P30CA023108-37) and (P30GM103415-15).

Studies involving human subjects were conducted according to the principles expressed in the Declaration of Helsinki. Studies were approved by Dartmouth College Institutional Review Board and the Committee for the Protection of Human Subjects (CPHS). Written informed consent was obtained before surgery from HIV-negative women undergoing hysterectomies at Dartmouth-Hitchcock Medical Center (Lebanon, NH). Trained pathologists selected tissue samples from the EM, CX, and ECX, free of pathological lesions and distant from the sites of pathology. Blood samples were obtained from volunteer healthy donors recruited at Dartmouth Hitchcock Medical Center. Blood donors were anonymous. Freshly isolated EM, CX, and ECX tissues from the operating room were transferred to Pathology for isolation and classification prior to transfer to our laboratory in separate, sterile polypropylene tubes.
1. Enzymatic Digestion of Tissues
NOTE: Use sterile materials and work in a biological safety cabinet.
<ol>
	<li>Prepare a modified Hank&#39;s Balanced Salt Solution (HBSS) using 1x HBSS with phenol red, 100 U/mL penicillin-streptomycin, 0.35 g/L NaHCO3, and 20 mM HEPES.</li>
	<li>Prepare the enzymatic cocktail (modified HBSS, 450 U/mL collagenase IV, 0.01% [wt/vol] deoxyribonuclease I, and 2 mg/mL D-glucose). Pass the solution through a sterile 0.22 &#181;m filter. Ideally, prepare the digestion enzyme on the day of use, but it can be frozen at -20 &#176;C for storage. Avoid repeated freezing and thawing cycles.</li>
	<li>Rinse the tissue with modified HBSS and place in a 100 x 15 mm2 Petri dish. Adjust the Petri dish size based on the amount of tissue available and the volume of enzyme cocktail used (see step 1.4.2 below). 
	NOTE: Tissues vary in size depending on the surgical sample obtained from the pathology, ranging from 1-10 g.</li>
	<li>Physical processing with disposable scalpel and forceps:
	<ol>
		<li>Add approximately 3 mL of digestion enzyme cocktail to the tissue to prevent drying while cutting.</li>
		<li>Use forceps to stabilize the tissue and mince with a scalpel to increase the surface area of the tissue exposed to the digestion enzyme cocktail. Cut the tissue into small pieces (&#60; 2 mm on a side). Add sufficient digestion enzyme cocktail to cover all tissue pieces (5 mL/g of tissue). Place the lid on the Petri dish.</li>
		<li>Incubate at 37 &#176;C with 5% CO2 on a rotator at approximately 80 rpm for 45 min. Ensure that the rotator speed thoroughly mixes the digestion enzyme cocktail without spilling or producing bubbles.</li>
		<li>Visualize the tissue preparation with an inverted light microscope with the 4X and 10X objectives. 
		NOTE: After digestion, small tissue fragments and a mixed cell suspension including visible epithelial glands should be present (see Figure 1A).</li>
	</ol>
	</li>
</ol>
2. Physical Separation of Single Cells
<ol>
	<li>In a sterile environment, place a taut 250 &#181;m mesh with holder into a 150 x 15 mm2 Petri dish. Ensure that the mesh is about 0.5 cm above the surface of the Petri dish.</li>
	<li>Wet the mesh with 2 mL of modified HBSS and transfer the digested tissue onto the mesh.</li>
	<li>Using the flat surface of a 10-mL syringe plunger, gently but firmly, grind the digested minced tissue through the mesh. 
	Caution: Grinding too aggressively will damage the mesh and increase cell mortality. This step will separate the epithelial cells and stromal cells (including immune cells) from the connective tissue and mucus.</li>
	<li>Once the tissue fragments have been thoroughly dispersed, elevate the mesh and the holder 2-3 cm above the Petri dish and rinse with 3 mL of modified HBSS to recover additional cells.</li>
	<li>Collect the cell suspension. Place the 20 &#181;m mesh with holder in a Petri dish and wet with 2 mL of modified HBSS. Pour the cell suspension through the mesh holding the mesh 2-3 cm above the Petri dish, and rinse with 6&#160;mL of modified HBSS. 
	NOTE: This step will separate the epithelial sheets, which are retained on the top, from single cells that pass through the mesh. The flow-through is a mixed cell suspension, which includes immune cells and stromal fibroblasts.</li>
	<li>Collect the mixed cell suspension and centrifuge at 500 x g for 10 min. Aspirate the liquid and resuspend the pellet in 4 mL of modified HBSS for density gradient centrifugation.</li>
	<li>Layer the mixed cell suspension over 3 mL of polysucrose solution and centrifuge at room temperature at 500 x g for 30 min with no brake. Collect the white band from the top of the polysucrose solution and wash with PBS. 
	NOTE: This fraction is the enriched mixed cell suspension, which is rich in immune cells and contains some stromal fibroblasts.</li>
</ol>
3. Dead Cell Removal
NOTE: If a large percentage of dead cells (&#62; 20%) are present in the enriched mixed cell suspension after density gradient centrifugation, dead cell removal with magnetic beads is recommended. Dead cells need to be removed since they may interfere with the positive magnetic bead selection process.
<ol>
	<li>Count the cells using a hemocytometer on a light microscope with the 10X objective. Spin down all the cells in the enriched mixed cell suspension (500 x g, 10 min, 4 &#176;C). Completely aspirate and discard the supernatant. Add 100 &#181;L of dead cell removal beads per 1 x 107 total cells (live and dead), according to the manufacturer&#39;s instructions. Incubate at room temperature for 15 min.</li>
	<li>Place a 30 &#181;m filter on the magnetic column, apply the cell suspension and allow the bead-labeled cells to fall through the magnetic column.</li>
	<li>Rinse the column with dead cell removal buffer as recommended (3 mL). Collect the flow-through containing the live cells. The range of cell recovery after dead cell removal is presented in Figure 1B. 
	NOTE: These cells can be used to perform flow cytometry staining for phenotypical studies (section 5) or continue with DC isolation (section 4).</li>
</ol>
4. DC Purification by Positive Magnetic Bead Selection
<ol>
	<li>Following dead cell removal (step 3.2), spin the cells down, remove the supernatant, and resuspend the cell pellet in 80 &#181;L magnetic selection buffer per 1 x 107 cells (PBS, 0.5% heat-inactivated human AB serum (HS), 2 mM EDTA).</li>
	<li>For positive selection of DC populations, add CD1a or CD14 magnetic beads according to the manufacturer&#39;s instructions (20 &#181;L magnetic beads per 1 x 107 cells). Incubate for 15 min at 4&#176;C.</li>
	<li>Wash the cells with the magnetic selection buffer, spin down, and remove buffer completely. Resuspend the cells in a minimum of 500 &#181;L of magnetic selection buffer.</li>
	<li>Attach the column to the magnet and add a 30 &#181;m filter on the top of the column to retain any remaining cell clumps. Rinse the filter and column with magnetic selection buffer as recommended.</li>
	<li>Apply the cell suspension to the column. Rinse 3 times with magnetic selection buffer.</li>
	<li>Transfer the column to a 15-mL tube, add 5 mL of magnetic selection buffer, and apply the plunger to release the selected cells from the column.</li>
	<li>To increase the purity, repeat steps 4.4-4.6 with the recovered cells using a new column. The range of cell recovery after magnetic bead selection is presented in Figure 1C.</li>
</ol>
5. Assessment of Cell Purity: Flow Cytometry and Microscopy
<ol>
	<li>Antibody staining for flow cytometry:
	<ol>
		<li>Resuspend up to 1 x 106 cells in 100 &#181;L of PBS containing 20% heat-inactivated HS to block Fc receptors, and incubate for 10 min on ice.</li>
		<li>Add the desired fluorescently labeled antibodies and the reagent for identification of dead cells (for an example, see section 6.5). Set fluorescence minus one (FMO) controls to establish the gates, and use compensation beads to prepare single stain controls for compensation. Examples of antibodies used are given in Table 1. Incubate for 20 min at 4 &#176;C, protected from light.</li>
		<li>Wash the cells with 2 mL of magnetic selection buffer. 
		NOTE: Presence of EDTA is important to avoid the formation of cell clumping for flow cytometric analysis. Spin down and discard the supernatant.</li>
		<li>Fix the cells by adding 100 &#181;L of 2% paraformaldehyde solution per tube.</li>
		<li>Analyze the samples in by a flow cytometer10,11.</li>
	</ol>
	</li>
	<li>Spin the column (e.g., cytospin) and use Giemsa staining to analyze morphology:
	<ol>
		<li>Resuspend the cells in 200 &#181;L of magnetic selection buffer.</li>
		<li>Load in a spin column and spin for 5 min. Let the slide dry completely.</li>
		<li>Fix the slide by immersion in 100% methanol for 2 min. Rinse the slide with deionized water and allow it to air dry.</li>
		<li>Perform a standard Wrights-Giemsa staining. Cover the cells with Eosin and incubate for 10 min, rinse with deionized water, and let it dry. Cover the cells with Wrights-Geimsa stain, incubate for 1 min, rinse with deionized water, and air dry.</li>
		<li>Examine the preparations with an inverted microscope (Figure 2A, 40X objective).</li>
	</ol>
	</li>
</ol>
6. Allogeneic Stimulation Assay to Assess DC Cell Function
<ol>
	<li>Thaw the frozen peripheral blood mononuclear cells (PBMCs)
	<ol>
		<li>Warm the cell culture media with 10% HS to 37 &#176;C.</li>
		<li>Thaw the PBMCs by placing the cryotube in a 37 &#176;C water bath, until a small amount of frozen cell media remains in the cryotube. In a sterile environment, transfer the contents of the cryotube to 10 mL of pre-warmed cell culture media with 10% HS in a 15-mL tube.</li>
		<li>Centrifuge at ~ 500 x g for 7 min. Remove the media, disperse the pellet, resuspend the PBMCs in 10 mL of cell culture media with 10% HS, and centrifuge the cells.</li>
		<li>Repeat the wash step a third time. Count the cells.</li>
	</ol>
	</li>
	<li>Na&#239;ve T-cell selection:
	<ol>
		<li>Use a negative selection magnetic antibody cell selection kit to isolate na&#239;ve T-cells according to the manufacturer&#39;s protocol.</li>
		<li>After isolation, check the purity using CD45RA and CCR7 antibodies. 
		NOTE: Typical purity is &#62; 99% na&#239;ve T-cells.</li>
	</ol>
	</li>
	<li>Na&#239;ve T-cell proliferation dye staining:
	<ol>
		<li>Prepare 600 &#181;L of 2x solution of cell proliferation dye, protecting the dye from exposure to light.</li>
		<li>Wash the na&#239;ve T-cells 2x with PBS. Resuspend cells in 500 &#181;L PBS.</li>
		<li>While gently vortexing the cells in the biosafety cabinet, add 500 &#181;L of proliferation dye to the na&#239;ve T-cells.</li>
		<li>Incubate the cells for 10 min at 37&#176;C.</li>
		<li>Stop the cell labeling by adding 5 mL of cell culture media with 10% HS to the cells. Incubate on ice for 5 min, protected from light.</li>
		<li>Centrifuge the cells at ~ 500 x g for 7 min, aspirate the media, and resuspend the cells in 4 &#176;C of cell culture media with 10% HS. Repeat two more times for a total of 3 washes. Take an aliquot of cells to count prior to the last centrifugation. 
		NOTE: Typically, final na&#239;ve T-cell numbers will range between 5% and 15% of the initial PBMC cell count.</li>
		<li>Resuspend the cells with the cell culture media with 10% HS at the desired cell concentration.</li>
	</ol>
	</li>
	<li>DC-allogeneic na&#239;ve T-cell co-culture:
	<ol>
		<li>Plate the isolated DCs and na&#239;ve T-cells in a 96-well, round-bottom plate, at a 1:15 ratio of DCs to T-cells. Ensure that the optimal number for DCs ranges between 3,000 and 5,000 cells/well.</li>
		<li>Centrifuge the plate at ~ 500 x g for 3 min to ensure the cells are located at the center of the well. Cell-to-cell contact is important for proper signaling to occur.</li>
		<li>Incubate at 37 &#176;C for 6 days. Monitor the cluster of cells, which appear in the wells as proliferation occurs, with microscopy (Figure 3A).</li>
	</ol>
	</li>
	<li>Flow cytometry staining to assess proliferation:
	<ol>
		<li>After 6 days, cells can be examined by flow cytometry. Stain the co-culture for flow cytometry.</li>
		<li>Prepare a 2x solution of yellow viability dye (maximum emission 572 nm) in PBS. 
		NOTE: The use of PBS without serum is important, as serum proteins interfere with the dye).</li>
		<li>Centrifuge the plate with cells at ~ 500 x g for 5 min.</li>
		<li>Remove 100 &#181;L of supernatant. 
		NOTE: At this point collect and store the supernatant for separate analysis of soluble factors.</li>
		<li>Wash the cells two times with PBS: add 150 &#181;L/well of PBS, centrifuge at ~ 500 x g for 5 min, remove 150 &#181;L of supernatant with a pipette. Ensure that there is 50 &#181;L of supernatant remaining in each well.</li>
		<li>Add 50 &#181;L of 2x viability dye to each well. Incubate at room temperature for 10 min, protected from light.</li>
		<li>During the incubation, prepare a master mixture of desired antibody markers. For example: CD3 APC/Cy7, CD4 PE, CD8 FITC, etc.</li>
		<li>Add the antibody mixture to each well. Incubate at room temperature for 10 min, protected from light.</li>
		<li>Wash the cells 2x with PBS + 2% HS: add 150 &#181;L/well of PBS + 2% HS. Centrifuge at ~ 500 x g for 5 min. Remove 150 &#181;L of supernatant with a pipette.</li>
		<li>Fix the cells by adding 100 &#181;L/well of 2% para-formaldehyde. If needed: transfer the cells and media to polypropylene flow cytometry tubes. Tubes can be kept at 4 &#176;C and protected from the light, until the flow cytometric analysis.</li>
		<li>Read tubes in a flow cytometer.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wira, C. R., Rodriguez-Garcia, M., Patel, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sex+hormones+in+immune+protection+of+the+female+reproductive+tract.">The role of sex hormones in immune protection of the female reproductive tract.</a> Nat Rev Immunol. 15 (4), 217-230 (2015).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392 (6673), 245-252 (1998).</li><li>Schlitzer, A., McGovern, N., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+monocyte-derived+cells:+Two+complementary+and+integrated+functional+systems.">Dendritic cells and monocyte-derived cells: Two complementary and integrated functional systems.</a> Semin Cell Dev Biol. 41, 9-22 (2015).</li><li>Tagliani, E., Erlebacher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+function+at+the+maternal-fetal+interface.">Dendritic cell function at the maternal-fetal interface.</a> Expert Rev Clin Immunol. 7 (5), 593-602 (2011).</li><li>Kaldensjo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intraepithelial+and+stromal+Langerin+and+CCR5+positive+cells+in+the+human+endometrium:+potential+targets+for+HIV+infection.">Detection of intraepithelial and stromal Langerin and CCR5 positive cells in the human endometrium: potential targets for HIV infection.</a> PLoS One. 6 (6), e21344 (2011).</li><li>Schulke, L., Manconi, F., Markham, R., Fraser, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+dendritic+cell+populations+during+the+normal+menstrual+cycle.">Endometrial dendritic cell populations during the normal menstrual cycle.</a> Hum Reprod. 23 (7), 1574-1580 (2008).</li><li>Ballweber, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaginal+langerhans+cells+nonproductively+transporting+HIV-1+mediate+infection+of+T+cells.">Vaginal langerhans cells nonproductively transporting HIV-1 mediate infection of T cells.</a> J Virol. 85 (24), 13443-13447 (2011).</li><li>Hladik, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+events+in+establishing+vaginal+entry+and+infection+by+human+immunodeficiency+virus+type-1.">Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1.</a> Immunity. 26 (2), 257-270 (2007).</li><li>Duluc, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+human+vaginal+APC+subsets+in+directing+T-cell+responses.">Functional diversity of human vaginal APC subsets in directing T-cell responses.</a> Mucosal Immunol. 6 (3), 626-638 (2013).</li><li>Herzenberg, L. A., Tung, J., Moore, W. A., Parks, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+flow+cytometry+data:+a+guide+for+the+perplexed.">Interpreting flow cytometry data: a guide for the perplexed.</a> Nat Immunol. 7 (7), 681-685 (2006).</li><li>Juno, J. A., Boily-Larouche, G., Lajoie, J., Fowke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation,+and+flow+cytometric+analysis+of+human+endocervical+samples.">Collection, isolation, and flow cytometric analysis of human endocervical samples.</a> J Vis Exp. (89), (2014).</li><li>Givan, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+leukocytes+in+the+human+female+reproductive+tract:+comparison+of+fallopian+tube,+uterus,+cervix,+and+vagina.">Flow cytometric analysis of leukocytes in the human female reproductive tract: comparison of fallopian tube, uterus, cervix, and vagina.</a> Am J Reprod Immunol. 38 (5), 350-359 (1997).</li><li>Rodriguez-Garcia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+from+the+human+female+reproductive+tract+rapidly+capture+and+respond+to+HIV.">Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV.</a> Mucosal Immunol. 10 (2), 531-544 (2017).</li><li>Rodriguez-Garcia, M., Barr, F. D., Crist, S. G., Fahey, J. V., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+susceptibility+to+HIV+infection+of+CD4++Th17+cells+in+the+human+female+reproductive+tract.">Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract.</a> Mucosal Immunol. 7 (6), 1375-1385 (2014).</li><li>Shen, Z., Rodriguez-Garcia, M., Ochsenbauer, C., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+immune+cells+and+infection+by+HIV+in+human+ovarian+tissues.">Characterization of immune cells and infection by HIV in human ovarian tissues.</a> Am J Reprod Immunol. , (2017).</li></ol>

The authors have nothing to disclose

Mucosal DCs are a rare cell population strongly influenced by the tissue environment, which changes their phenotype and function once they enter the tissues3. While blood derived DCs are a very useful model, they do not fully represent the diversity of DC populations found in tissues. Therefore, to understand the unique characteristics of mucosal DCs, isolation of primary cells from tissues is necessary. Isolation of DCs from different anatomical sites in the FRT offers the opportunity to study DC function in vitro and compare between DC subsets and anatomical locations.
Here we provide a protocol that allows the purification of DCs from human FRT tissues for in vitro assessment of functional characteristics. Since DCs are found at mucosal sites in low numbers, we developed a protocol to enrich the tissue cell preparation by density gradient centrifugation and dead cell removal prior to cell isolation with magnetic beads. Proper removal of dead cells is key, as they will interfere with bead isolation. This technique provides high purity of positively isolated cells (&#8776; 90%), in a relatively short amount of time (4-6 h), and without the need for overnight incubation, in vitro cell culture, or migration prior to isolation, each of which can activate DCs and alter their phenotypic characteristics. Our protocol does not trigger cell activation, as measured by the lack of up-regulation of maturation markers (CD86, HLA-DR, CD83)13. Another advantage is the use of a non-proteolytic enzymatic digestion, which does not cleave surface markers and allows for immediate cell isolation or phenotypical analysis following tissue digestion14.
Critical to this protocol is the generation of a single cell suspension to avoid the blockage of the magnetic columns. To ensure this, dead cells must be removed prior to the magnetic cell selection, filters need to be used on top of the magnetic columns, and tissues larger than 3 g should be divided between two columns. The use of a second column after the first round of purification is key to high cell purity.
One limitation of the isolation method is the inherent low number of DCs in FRT tissues, which usually does not allow for good cell recovery of tissues smaller than 1 g. Under these circumstances, however, phenotypical analyses can still be performed using the enriched mixed cell suspension. This is possible due to the non-proteolytic digestion method used, which does not cleave surface markers and allows for immediate analysis of cell phenotype after tissue digestion14. Additionally, the patient age can be a factor that influences the number of cells recovered.
Using this protocol, we have previously demonstrated that CD1a+ isolation provides a phenotypically more homogeneous population than CD14+ selection13; however, CD1a+ DCs are difficult to recover from CX and ECX. While CD14 can also be expressed on macrophages, we found that the FRT CD14+ isolated population is rich in DCs, based on co-expression of DC markers and their ability to stimulate allogeneic na&#239;ve T-cells, which is a hallmark function of DCs13.
Since the number of DCs recovered is generally low (&#60; 100,000 total cells), functional studies need to be optimized for low cell numbers. Here we show the optimization of an allogeneic stimulation assay, which can be performed with only 3,000 DCs/well. We have carried out other function studies with these cells, including hormonal responsiveness, cytokine, and antimicrobial peptide production upon HIV-stimulation and HIV-capture by FRT DCs13. Once the DCs are isolated and plated, assays should be performed within 24 h, as cell viability decreases after that time.
This protocol can be adapted to isolate different DC subsets, or other cell types by selecting the proper markers coupled to the magnetic beads and combining sequential positive and negative bead selection to remove unwanted cell types. One caution, though, is that with every round of selection some percentage of cells will be lost, so for the isolation of FRT DCs, which are already rare, and the tissue size is generally small, multiple rounds of selection with different markers (and the necessary washes associated) are not desirable. Moreover, this protocol can be adapted to isolate other immune cells or DCs from other tissues14,15.

The FRT has the dual function of protecting against pathogens while allowing implantation and pregnancy1. To accomplish this, the FRT is compartmentalized, with each anatomical region displaying unique histological, immunological, and functional characteristics1.
DCs present at mucosal surfaces make contact with microbes in the lung, the gut, and the genital tract, and provide immune surveillance for potential pathogens2. DCs have the unique ability to prime na&#239;ve T-cells and trigger adaptive immune responses3. DCs in the FRT are also specialized to tolerate foreign antigens, such as those found in sperm and the developing fetus, to allow successful pregnancy4. Therefore, depending on the location, the DC phenotype and function will be distinct. It is known that DCs are strongly influenced by the tissue environment, such that their number, phenotype, and functions are modified by the tissue environment in which they reside3. Therefore, to understand the role that FRT DCs play in infectious diseases, pregnancy, and cancer in the FRT, resident DCs need to be studied, since blood derived DC models are inadequate to address the regulatory complexities found in FRT tissues.
The characterization of human tissue resident DCs is challenging due to the low numbers of cells present in mucosal tissues and the difficulty obtaining human tissue samples. DCs have been studied in the FRT using immunohistochemistry5,6, which informs about the cell location within the tissue, but precludes functional studies, and is limited in the number of identifying cell markers that can be analyzed at once. Moreover, single cell isolation protocols for flow cytometric analysis have been developed7,8,9. Some of these protocols take advantage of the migratory capacity of DCs to isolate those cells that migrate. These methods usually require overnight incubations and selection of activated DCs, but do not allow for the study of DCs in the steady state.
Here, using hysterectomy specimens, we optimized a protocol to isolate DCs from different anatomical sites in the FRT, the endometrium (EM), the endocervix (CX), and the ectocervix (ECX), that enables both phenotypical and functional analyses. Using a non-proteolytic enzymatic digestion protocol, we can proceed immediately after tissue digestion to cell isolation and flow cytometric characterization without cell activation. Using multicolor flow cytometry and adapting functional studies for low numbers of cells, we can identify and characterize rare subsets of DCs in the different sites of the FRT.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:203px;">Hyclone</td>
			<td style="width:119px;">SH30015.03</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Hyclone</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I</td>
			<td>Worthington Biochemical</td>
			<td>LS002140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Sigma Aldritch</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 um Stericup 500 mL&#160; filter</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm polystyrene petri dish</td>
			<td>Fisherbrand</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150mm x 15mm polystyrene petri dish</td>
			<td>Fisherbrand</td>
			<td>FB0875714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150mm x 25mm polystyrene dish</td>
			<td>Corning</td>
			<td>430599</td>
			<td>Treated cell culture dish</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Isotemp Incubator</td>
			<td>Fisher Scientific</td>
			<td>FICO3500TABB</td>
			<td>5.0% CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">American Rotator V</td>
			<td>American DADE</td>
			<td>R4140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 um nylon mesh</td>
			<td>Sefar</td>
			<td>03-250/50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 um nylon mesh</td>
			<td>Sefar</td>
			<td>03-20/14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beckman GS-6R Centrifuge</td>
			<td>Beckman</td>
			<td>358702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human AB Serum</td>
			<td>Valley Biomedical</td>
			<td>HP1022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma Aldritch</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution (PBS)</td>
			<td>National Diagnostics</td>
			<td>CL-253</td>
			<td>pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pre-separation filter (30um)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadro MACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS multi-stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>USB</td>
			<td>15694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD1a Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-051-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD14 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eFluor 670 cell proliferation dye</td>
			<td>eBiosciences</td>
			<td>65-0840-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well round bottom plate</td>
			<td>Falcon</td>
			<td>9/8/2866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie yellow viability dye</td>
			<td>Biolegend</td>
			<td>423104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3 APC/Cy7, anti-human</td>
			<td>Tonbo Biosciences</td>
			<td>25-0038-T100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4 PE, anti-human</td>
			<td>eBiosciences</td>
			<td>12-0048-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a FITC, anti-human</td>
			<td>Tonbo Biosciences</td>
			<td>35-0086-T100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gallios Flow Cytometer</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>B43618</td>
			<td>10 color, VBR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSquant Analyzer 10</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-343</td>
			<td>8 color, VBR</td>
		</tr>
	</tbody>
</table>

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.

Following tissue digestion, the release of epithelial sheets and glands can be observed, as shown in Figure 1A; this is a positive control that indicates the enzymatic digestion was successful. The number of total viable cells and DCs recovered per gram of tissue are also shown in Figure 1B and Figure 1C, respectively. Immune cells represent between 5-30% of the stromal cells prior to density centrifugation12,13. Figure 2 shows the morphology (Figure 2A) and purity (Figure 2B, C) of the isolated DCs. When two rounds of selection are performed as indicated in the protocol, the expected purity ranges between 85-92% (Figure 2C). The number of recovered cells is variable and depends on the initial tissue size and donor variability. Characterization of the isolated cells have been described13. Figure 3 shows a representative allogeneic stimulation assay to assess DC functionality. Figure 3A shows the characteristic T-cell clumps that appear when proliferation occur, and Figure 3B shows the proliferation quantification by flow cytometry. Other functional assays, such as viral capture or cytokine and chemokine production can also be performed13. Figure 4 shows the gating strategy to identify different DCs populations in the distinct FRT anatomical compartments after flow-cytometric analysis of enriched mixed cell suspensions (Figure 4A). The titration of antibodies and negative controls are important, as DCs and macrophages display high autofluorescence. FMO controls are shown in Figure 4B. Figure 4C shows examples of how to identify specific DC subsets, such as CD1c+, CD207+, or CD103+ DCs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57100/57100fig1.jpg" /> 
Figure 1: Tissue processing and cell availability. (A) Representative image of epithelial sheets and glands released after tissue digestion. (B) Range of total number of viable cells recovered after tissue processing and dead cell removal in each FRT compartment: endometrium (EM), endocervix (CX), and ectocervix (ECX). Every dot represents cells from a different subject. (C) Number of DCs recovered per gram of tissue after magnetic bead isolation. B and C are adapted from13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57100/57100fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57100/57100fig2.jpg" /> 
Figure 2: Morphology and purity of the isolated cells. (A) Dendritic morphology of the isolated cells determined by microscopy following Giemsa staining. (B) Expression of phenotypic markers before and after bead isolation, as determined by flow cytometry. (C) Representative purity obtained after CD1a+ and CD14+ bead isolation. Adapted from13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57100/57100fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57100/57100fig3.jpg" /> 
Figure 3: Proliferation assay. (A) Representative microscopy images of T-cell cluster formation during proliferation: phase-contrast (left), proliferation dye staining (middle), and overlay (right). (B) Representative example of induction of proliferation after co-culture of isolated endometrial CD1a+ or CD14+ DCs and allogeneic na&#239;ve T-cells obtained from frozen PBMCs. B is adapted from13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57100/57100fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57100/57100fig4.jpg" /> 
Figure 4: Phenotypical characterization of DCs in the enriched mixed cell suspensions from the FRT. (A) Gating strategy for the identification of DCs in the mixed cell suspension. Each gated population in multicolor plots is shown in the next panel. Contour plots show the CD11c+ CD11b+ and CD11c+ CD11b- gates, respectively. Markers that identify unwanted cells can be stained using the same channel to exclude them from the analysis; for example, dead cells, CD3+, and CD19+ cells. (B) FMO controls to establish appropriate gates. (C) Discrimination of DC subsets following the gating strategy. Contour plots were chosen instead of multicolor plots to more accurately represent the population distribution when the cell numbers were low10. Low cell numbers are expected at the end of the gating strategy, as tissue DCs are rare populations. Adapted from13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57100/57100fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell marker</td>
			<td>Fluorochrome</td>
			<td>Clone</td>
			<td>Concentration</td>
			<td>Amount of antibody per sample</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;(in 100 &#181;l of buffer)</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>vioblue450</td>
			<td>HI30</td>
			<td>1.0 &#181;g/5&#181;l</td>
			<td>0.4 &#181;g</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>PE</td>
			<td>ICRF44</td>
			<td>1.0 &#181;g/5&#181;l</td>
			<td>0.4 &#181;g</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>PerCp/Cy5.5</td>
			<td>Bu15</td>
			<td>200 &#181;g/ml</td>
			<td>0.4 &#181;g</td>
		</tr>
		<tr>
			<td>CCR5</td>
			<td>PE-Cy7</td>
			<td>2D7</td>
			<td>0.25 &#181;g/5&#181;l</td>
			<td>0.1 &#181;g</td>
		</tr>
		<tr>
			<td>CCR7</td>
			<td>PE-Cy7</td>
			<td>REA108</td>
			<td>99 &#181;g/ml</td>
			<td>0.2 &#181;g</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>BV-570</td>
			<td>L243</td>
			<td>100 &#181;g/ml</td>
			<td>0.2 &#181;g</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>FITC</td>
			<td>AC122</td>
			<td>82.5 &#181;g/ml</td>
			<td>0.16 &#181;g</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>APC</td>
			<td>UCHT1</td>
			<td>16 &#181;g/ml</td>
			<td>0.03 &#181;g</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>viogreen</td>
			<td>REA614</td>
			<td>137 &#181;g/ml</td>
			<td>0.27 &#181;g</td>
		</tr>
		<tr>
			<td>CD163</td>
			<td>APC</td>
			<td>GHI/61</td>
			<td>80 &#181;g/ml</td>
			<td>0.16 &#181;g</td>
		</tr>
		<tr>
			<td>CD207</td>
			<td>APC</td>
			<td>1OE2</td>
			<td>200 &#181;g/ml</td>
			<td>0.4 &#181;g</td>
		</tr>
		<tr>
			<td>CD1a</td>
			<td>FITC</td>
			<td>HI149</td>
			<td>0.5 &#181;g/5&#181;l</td>
			<td>0.2 &#181;g</td>
		</tr>
		<tr>
			<td>CD1a</td>
			<td>AF-700</td>
			<td>HI149</td>
			<td>0.5 mg/ml</td>
			<td>1.0 &#181;g</td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>PE-Cy7</td>
			<td>B-Ly7</td>
			<td>0.25 &#181;g/5&#181;l</td>
			<td>0.1 &#181;g</td>
		</tr>
		<tr>
			<td>CD83</td>
			<td>PE</td>
			<td>HB15e</td>
			<td>0.25 &#181;g/5&#181;l</td>
			<td>0.1 &#181;g</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>APC-Fire</td>
			<td>63D3</td>
			<td>400 &#181;g/ml</td>
			<td>0.8 &#181;g</td>
		</tr>
		<tr>
			<td>CD209</td>
			<td>FITC, PE, APC</td>
			<td>120507</td>
			<td>1.25 &#181;g/0.25ml</td>
			<td>0.01 &#181;g</td>
		</tr>
		<tr>
			<td>Zombie yellow viability dye</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7AAD</td>
			<td></td>
			<td></td>
			<td>0.1mg/ml</td>
			<td>0.2ug</td>
		</tr>
	</tbody>
</table>
Table 1: Example of antibodies for the characterization of DCs in the FRT by flow cytometry.

Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

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

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 ...

isolation-dendritic-cells-from-human-female-reproductive-tract-for

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Immunology and Infection

6.4K Views.  Geisel School of Medicine at Dartmouth. 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.

Video: Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 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 ...

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 ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...