Name: isolation of dendritic cells from the human female reproductive tract for phenotypical and functional studies
Uploaded: 2018-03-13T15:00:00.000+00:00
Duration: 12 min 32 s
Description: Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

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...

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><tbody><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Hyclone</td><td>SH30015.03</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>HEPES (1M)</td><td>Hyclone</td><td>15630-080</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C5138</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical</td><td>LS002140</td><td></td></tr><tr><td>D-glucose</td><td>Sigma Aldritch</td><td>50-99-7</td><td></td></tr><tr><td>0.22 um Stericup 500 mL&amp;nbsp; filter</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>100mm x 15mm polystyrene petri dish</td><td>Fisherbrand</td><td>FB0875712</td><td></td></tr><tr><td>150mm x 15mm polystyrene petri dish</td><td>Fisherbrand</td><td>FB0875714</td><td></td></tr><tr><td>150mm x 25mm polystyrene dish</td><td>Corning</td><td>430599</td><td>Treated cell culture dish</td></tr><tr><td>Isotemp Incubator</td><td>Fisher Scientific</td><td>FICO3500TABB</td><td>5.0% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>American Rotator V</td><td>American DADE</td><td>R4140</td><td></td></tr><tr><td>250 um nylon mesh</td><td>Sefar</td><td>03-250/50</td><td></td></tr><tr><td>20 um nylon mesh</td><td>Sefar</td><td>03-20/14</td><td></td></tr><tr><td>Beckman GS-6R Centrifuge</td><td>Beckman</td><td>358702</td><td></td></tr><tr><td>X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>Human AB Serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma Aldritch</td><td>10771</td><td></td></tr><tr><td>Phosphate Buffer Solution (PBS)</td><td>National Diagnostics</td><td>CL-253</td><td>pH 7.4</td></tr><tr><td>Dead Cell Removal Kit</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></td></tr><tr><td>Pre-separation filter (30um)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>LS column</td><td>Miltenyi Biotec</td><td>130-042-410</td><td></td></tr><tr><td>Quadro MACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>MACS multi-stand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>EDTA</td><td>USB</td><td>15694</td><td></td></tr><tr><td>CD1a Microbeads, human</td><td>Miltenyi Biotec</td><td>130-051-001</td><td></td></tr><tr><td>CD14 Microbeads, human</td><td>Miltenyi Biotec</td><td>130-050-201</td><td></td></tr><tr><td>eFluor 670 cell proliferation dye</td><td>eBiosciences</td><td>65-0840-85</td><td></td></tr><tr><td>96 well round bottom plate</td><td>Falcon</td><td>9/8/2866</td><td></td></tr><tr><td>Zombie yellow viability dye</td><td>Biolegend</td><td>423104</td><td></td></tr><tr><td>CD3 APC/Cy7, anti-human</td><td>Tonbo Biosciences</td><td>25-0038-T100</td><td></td></tr><tr><td>CD4 PE, anti-human</td><td>eBiosciences</td><td>12-0048-42</td><td></td></tr><tr><td>CD8a FITC, anti-human</td><td>Tonbo Biosciences</td><td>35-0086-T100</td><td></td></tr><tr><td>Gallios Flow Cytometer</td><td>Beckman Coulter Life Sciences</td><td>B43618</td><td>10 color, VBR</td></tr><tr><td>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 centrif...

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

Research

JoVE Journal

Immunology and Infection

6.5K 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 Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

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 ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

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 ...

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance 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 ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

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

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Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation of Mouse Lung Dendritic Cells

Culture of myeloid dendritic cells from bone marrow precursors

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

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells