The National Institutes of Health (NIH R15 NS067536-01A1 to DC), the National Vulvodynia Association (award to DC), and Macalester College supported this work. CB received a fellowship from the Macalester College Beckman Scholars Program, funded by the Arnold and Mabel Beckman Foundation. BTF and TM are supported by JDRF 2-2011-662. We thank Dr. Jason Schenkel and Dr. Juliana Lewis for technical advice, and all current and former members of the Chatterjea lab for their help and support.

Note: 8-12 week old female ND4 Swiss Webster mice, conventionally housed with free access to food and water, were used for these studies. The experimental protocol used (B13S1) was approved by Macalester College&#39;s IACUC.
1. Sensitization and Challenge with Oxazolone
<ol>
	<li>Day 0
	<ol>
		<li>Prepare anesthesia chamber by adding 3 ml isoflurane to absorbent towels placed beneath mesh at the bottom of a 4 L lidded glass jar. For the ND4 female mice used here, time in the chamber is 30-60 sec&#160;until the subject is adequately anesthetized; optimize anesthetic administration for the mouse strain being used.</li>
		<li>Shave each anesthetized mouse&#39;s back and flank using an animal hair trimmer.</li>
	</ol>
	</li>
	<li>Day 1
	<ol>
		<li>Make a 2% solution of 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (Ox) into absolute ethanol, and incubate at 50 &#176;C for 15 min&#160;on a rotating plate in an incubator.</li>
		<li>Briefly anesthetize mice using isoflurane anesthesia as described in 1.1.1 and apply 100 &#181;l of 2% Ox to the shaved back in several serial applications of about 20 &#181;l&#160;each. Wait 5-10 sec&#160;between serial applications for the solution to dry.</li>
		<li>Take care to avoid spilling the 2% Ox solution on regions other than the back, and wait for the solution to dry between applications.</li>
	</ol>
	</li>
	<li>Days 5-7
	<ol>
		<li>Make a 1% solution of (Ox) in absolute ethanol, and incubate at 50 &#176;C for 15 min&#160;on a rotating plate in an incubator.</li>
		<li>Gently but firmly immobilize the mouse at the scruff of the neck and tail base with abdomen facing up and neck slightly tilted downward (similar to a hold for an intraperitoneal injection) and apply 100 &#181;l of 1% Ox to the shaved flank in several serial applications and taking the time to dry the skin afterwards as described above.</li>
		<li>For control mice, apply 100 &#181;l of absolute ethanol vehicle to the shaved flank.</li>
	</ol>
	</li>
</ol>
2. Flank Skin Harvest
<ol>
	<li>Euthanize mice using carbon dioxide inhalation, and carefully excise the desired area of flank skin (~25 mm x 25 mm of skin) with 10 cm surgical scissors.</li>
	<li>Using a clean, sharp surgical blade, scrape away the excess fat and connective tissue from the flank skin.</li>
	<li>Place 3 pieces of flank skin from 3 mice into a 50 ml conical tube containing 10 ml RT HBSS [with phenol red, without calcium or magnesium, 5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10% fetal bovine serum (FBS)].</li>
</ol>
3. Pre-digestion Tissue Washing
<ol>
	<li>Place tube containing skin fragments on a rotating plate for 30 min&#160;in a dry non-gassed incubator maintained at 37&#176;C.</li>
	<li>Vortex vigorously for 10 sec at the end of the incubation period.</li>
	<li>Strain the contents of the tube through a 70 &#181;m strainer to discard the wash buffer and collect the tissue. A single strainer can be reused throughout the entire procedure within a biological treatment group.</li>
	<li>Using a pair of forceps, transfer the flank skin from the strainer into a new 50 ml conical tube containing 10 ml of fresh, RT HBSS media (containing EDTA, HEPES, and FBS as described above).</li>
	<li>With a 12.5 cm pair of surgical dissecting scissors immersed into the tube, mince each piece of flank skin so that the average piece of tissue is approximately 2.2 mm x 2.2 mm in area.</li>
	<li>Place tube containing skin fragments on a rotating plate for 30 min&#160;in a dry non-gassed incubator maintained at 37&#176;C.</li>
	<li>Vortex vigorously for 10 sec at the end of the incubation</li>
</ol>
4. Collagenase Digestion of Skin
<ol>
	<li>Strain the contents of the tube through a 70 &#181;m strainer, collect and transfer the flank skin pieces into a new 50 ml conical tube containing 10 ml fresh, RT HBSS media supplemented with 0.7 mg/ml collagenase D.</li>
	<li>Place tube containing digestion medium and skin fragments on a rotating plate for 30 min&#160;in a dry non-gassed incubator maintained at 37&#176;C.</li>
	<li>Vortex contents of tube vigorously for 10 sec at the end of the incubation.</li>
	<li>Filter contents of tube through a 70 &#181;m strainer into a new 50 ml conical tube; keep the flow-through containing the digested tissue and discard the strainer and flank skin debris.</li>
	<li>Wash the flow-through in 50 ml of HBSS media, centrifuge for 5 min&#160;at 350 g, and decant the supernatant.</li>
</ol>
5. Density Gradient Centrifugation
<ol>
	<li>Using a serological pipette, add 3 ml RT 67% density gradient centrifugation media in 1X phosphate buffered saline (PBS) into a new 15 ml conical tube for each sample.</li>
	<li>Resuspend the cell pellet in 5 ml RT 44% density gradient centrifugation media in HBSS with phenol red.</li>
	<li>Gently layer 5 ml of the 44% density gradient centrifugation media containing the cells on top of the 67% density gradient centrifugation media sample using a serological pipette. Make sure to set the pipettor to the slowest speed, and place the pipette along the wall of the conical tube. Be careful not to shake, disrupt, or invert the gradient.</li>
	<li>Set acceleration to the lowest setting, disengage the brake, and centrifuge the gradient at 931 x g for 20 min&#160;at RT making sure that the centrifuge is balanced.</li>
	<li>After centrifugation, carefully remove the gradient from the centrifuge, and gently transport to the lab bench holding the tube upright. Using a 5 ml plastic transfer pipette, remove the cell layer at the interface of the 44% and 67% density gradient centrifugation media and transfer to a new 15 ml conical tube. If the interface is not visible, simply remove about 2 ml of liquid at the 67%-44% boundary.</li>
	<li>Fill the conical tube containing cells from the interface with 2% FBS in 1X ice-cold PBS, centrifuge cells for 5 min&#160;at 350&#160;x g, and decant the supernatant. 
	Note: This is the first time that the cells can be cooled/kept cold since digestion steps are at 37&#176;C and density gradient centrifugation media steps are at RT. Cells can be resuspended in a 1:1 Trypan blue dilution and counts and viability information can be obtained at this point.</li>
</ol>
6. Blocking and Staining for Flow Cytometric Analysis
<ol>
	<li>Block Fc&#947;RII/III receptors on cells to prevent non-specific antibody staining. For the experiments described here, make antibody master mixes in PBS with 2% FBS containing 1:100 dilution of unconjugated antibody against CD16/32 (2.4G2) to bind and block Fc&#947;RII/III receptors.</li>
	<li>Incubate blocked cells with appropriate master mixes of antibodies against CD3&#603; (145-2C11), CD4 (RM4-5), CD8&#945; (53-6.7), CD11b (M1/70), CD11c (N418), CD44 (IM7), CD45 (30-F11), CD45R (RA3-6B2), CD62L (MEL-14), and Ly-6G/Ly-6C (Gr-1) (RB6-8C5), as well as Brilliant Violet 510 live/dead stain to identify dead cells. Use all antibodies either at the dilution recommended by manufacturers or previously optimized by researchers. For the experiments described here, use all antibodies at 1:100 dilution.</li>
	<li>Wash cells and resuspend in 50 &#181;l 1X PBS staining buffer with 2% FBS. Keep on ice until fluorescence data can be acquired on a flow cytometer. 
	Note: It is important to have compensation parameters pre-optimized with a lymphoid tissue and a common cell surface antigen (like CD4 or CD8) before acquisition of fluorescence data.</li>
	<li>To increase the number of cells for downstream flow cytometry analysis, acquire data from entire samples of stained cells.</li>
</ol>
7. Analyze Flow Cytometry Data using Standard Methods to Accomplish the Steps Outlined Below
<ol>
	<li>First, gate on the lymphocyte region of the forward scatter (area) by side scatter (area) plot.</li>
	<li>Then, select single cells using the forward scatter (width) by side scatter (width), to avoid analyzing doublets. This can also be accomplished with a forward scatter height by side scatter height plot.</li>
	<li>Then, gate on lineage (CD11b, CD11c, and CD45R/B220)-negative and CD3-positive events, to exclude macrophages, dendritic cells, and B cells, respectively, and confine analyses to the T cell compartment, if the goal is, as it is here, to assess infiltrating T cells.</li>
	<li>Next, gate on live cells that exclude the live/dead stain.</li>
	<li>Finally, gate on the cell population of interest (see representative results section).</li>
</ol>

<ol>
	<li>Hay, R. J., 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=The+Global+Burden+of+Skin+Disease+in+2010:+An+Analysis+of+the+Prevalence+and+Impact+of+Skin+Conditions.">The Global Burden of Skin Disease in 2010: An Analysis of the Prevalence and Impact of Skin Conditions.</a> J. Invest. Dermatol. 134 (6), 1-8 (2013).</li><li>Honda, T., Egawa, G., Grabbe, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+immune+events+in+the+murine+contact+hypersensitivity+model:+toward+the+understanding+of+allergic+contact+dermatitis.">Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis.</a> J. Invest. Dermatol. 133 (2), 303-315 (2013).</li><li>Gudjonsson, J. E., Johnston, A., Dyson, M., Valdimarsson, H., Elder, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+psoriasis.">Mouse models of psoriasis.</a> J. Invest. Dermatol. 127 (6), 1292-1308 (2007).</li><li>Malachowa, N., Kobayashi, S. D., Braughton, K. R., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Innate+Immunity.">Mouse Models of Innate Immunity.</a> Mouse Models of Innate Immunity: Methods and Protocols. 1031, 109-116 (2013).</li><li>Mackay, L. K., 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=Cutting+Edge:+CD69+Interference+with+Sphingosine-1-Phosphate+Receptor+Function+Regulates+Peripheral+T+Cell+Retention.">Cutting Edge: CD69 Interference with Sphingosine-1-Phosphate Receptor Function Regulates Peripheral T Cell Retention.</a> J. Immun. 194, 2059-2063 (2015).</li><li>Schaerli, P., 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=A+skin-selective+homing+mechanism+for+human+immune+surveillance+T+cells.">A skin-selective homing mechanism for human immune surveillance T cells.</a> J. Exp. Med. 199 (9), 1265-1275 (2004).</li><li>Zaid, A., 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=Persistence+of+skin-resident+memory+T+cells+within+an+epidermal+niche.">Persistence of skin-resident memory T cells within an epidermal niche.</a> Proc. Natl. Acad. Sci. U. S. A. 111 (14), 5307-5312 (2014).</li><li>Harris, J. E., Harris, T. H., Weninger, W., Wherry, E. J., Hunter, C. A., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+vitiligo+with+focused+epidermal+depigmentation+requires+IFN-&#915;+for+autoreactive+CD8++T+cell+accumulation+in+the+skin.">A mouse model of vitiligo with focused epidermal depigmentation requires IFN-&#915; for autoreactive CD8+ T cell accumulation in the skin.</a> J. Invest. Dermatol. 132 (7), 1869-1876 (2012).</li><li>Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Single-Cell+Suspensions+from+Mouse+Spleen+with+the+gentleMACS+Dissociator.">Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator.</a> J. Vis. Exp. (22), e1029 (2008).</li><li>Nagao, K., 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=Murine+epidermal+Langerhans+cells+and+langerin-expressing+dermal+dendritic+cells+are+unrelated+and+exhibit+distinct+functions.">Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 3312-3317 (2009).</li><li>Pennartz, S., Reiss, S., Biloune, R., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+single-cell+suspensions+from+mouse+neural+tissue.">Generation of single-cell suspensions from mouse neural tissue.</a> J. Vis. Exp. (29), e1267 (2009).</li><li>Dudeck, A., 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=Mast+cells+are+key+promoters+of+contact+allergy+that+mediate+the+adjuvant+effects+of+haptens.">Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.</a> Immunity. 34 (6), 973-984 (2011).</li><li>Hershko, A. Y., 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=Mast+Cell+Interleukin-2+Production+Contributes+to+Suppression+of+Chronic+Allergic+Dermatitis.">Mast Cell Interleukin-2 Production Contributes to Suppression of Chronic Allergic Dermatitis.</a> Immunity. 35 (4), 562-571 (2011).</li><li>Huang, H. 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=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J. Biomed. Sci. 17 (36), (2010).</li><li>Tabatabaei, 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=Isolation+and+partial+characterization+of+human+amniotic+epithelial+cells:+The+effect+of+trypsin.">Isolation and partial characterization of human amniotic epithelial cells: The effect of trypsin.</a> Avicenna J Med. Biotechnol. 6 (1), 10-20 (2014).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> Eur. J. Microbiol. Immunol. 2, 112-120 (2012).</li><li>Gu, D., Fan, Q., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+population+analyses+during+skin+carcinogenesis.">Cell population analyses during skin carcinogenesis.</a> J. Vis. Exp. (78), e50311 (2013).</li><li>Broggi, M. A. S., Schmaler, M., Lagarde, N., Rossi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Murine+Lymph+Node+Stromal+Cells.">Isolation of Murine Lymph Node Stromal Cells.</a> J. Vis. Exp. (90), e51803 (2014).</li><li>Martinov, 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=Contact+hypersensitivity+to+oxazolone+provokes+vulvar+mechanical+hyperalgesia+in+mice.">Contact hypersensitivity to oxazolone provokes vulvar mechanical hyperalgesia in mice.</a> PloS one. 8 (10), e78673 (2013).</li><li>Katsares, V., 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=A+Rapid+and+Accurate+Method+for+the+Stem+Cell+Viability+Evaluation:+The+Case+of+the+Thawed+Umbilical+Cord+Blood.">A Rapid and Accurate Method for the Stem Cell Viability Evaluation: The Case of the Thawed Umbilical Cord Blood.</a> Lab. Med. 40 (9), 557-560 (2009).</li><li>Thomas, M. L., Lefran&#231;ois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+leucocyte-common+antigen+family.">Differential expression of the leucocyte-common antigen family.</a> Immunol. Today. 9 (10), 320-326 (1988).</li><li>Daley, J. M., Thomay, A. A., Connolly, M. D., Reichner, J. S., Albina, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Ly6G-specific+monoclonal+antibody+to+deplete+neutrophils+in+mice.">Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.</a> J. Leukoc. Biol. 83 (1), 64-70 (2008).</li></ol>

The authors have no financial or other interests to disclose.

Characterizing changes in skin-resident leukocytes in rodent models of skin diseases such as atopic dermatitis or psoriasis is important for understanding mechanistic connections between inflammatory cell influx and disease pathology. Here we describe an economical technique to isolate leukocytes from skin tissue with basic equipment found in most biomedical research labs. This relatively rapid technique avoids the use of expensive tissue dissociation machines and custom tubes and reagents, helping to conserve resources ...

Skin conditions ranging from contact dermatitis, eczema, psoriasis, cellulitis, fungal infections and abscesses to non-melanoma skin cancers were found to be among the 50 most prevalent diseases worldwide, and the fourth leading global cause of non-fatal diseases in 20101. Accordingly, the investigation of molecular and cellular mechanisms underlying diverse skin pathologies is a necessary and active area of research. Rodent models have been remarkably useful in the understanding of inflammatory skin conditions such as atopic dermatitis2, psoriasis3, or Staphylococcus aureus infection4. Inexpensive, efficient, and simple protocols for the enzymatic digestion of mouse skin tissue can provide preparations of cells that can be used for a variety of downstream applications to better understand the pathophysiology of skin diseases. Here, a simple and economical method is described for enzymatic digest of mouse skin tissue and isolation of skin infiltrating leukocytes that can be used for cell culture, in vivo adoptive transfer, flow cytometric analysis and sorting or gene expression studies. The overall goal of this procedure is to prepare a single cell suspension of skin-infiltrating leukocytes with high cell viability while minimizing costs typically associated with custom reagent kits and mechanical dissociators.
Existing skin tissue dissociation methods5-7 may result in low cell viability and surface marker integrity, or require custom enzyme kits and expensive tissue dissociation machines8-11. While the digestion of mouse ear skin tissue is reasonably prevalent12-13, digesting highly keratinized skin tissue (e.g. from the flank) can result in cell preparations contaminated with large amounts of non-cellular debris. In a recent study, Zaid and colleagues digested mouse flank skin for 90 min&#160;in 2.5 mg/ml dispase, followed by 45 min&#160;in 3 mg/ml&#160;collagenase7. In another study, these researchers used multiple incubations with a combined digestion of 2.5 hr, including the use of trypsin/EDTA, collagenase III, and dispase5. The use of trypsin is not recommended for enzymatic skin digestion, as treatment with trypsin from different manufacturers has been shown to measurably affect the integrity of cell surface proteins on mammalian cells14-15. Additionally, dispase can have significant effects on proliferative abilities of CD4 and CD8&#945; T cells and affect surface abundance of at least 20 molecules, including common T cell activation markers such as CD62L16. Other protocols use RPMI 1640 in the digestion medium6. However, the presence of Mg2+ and Ca2+ in RPMI can cause extensive cell aggregation17.
An ideal protocol for tissue dissociation should aim for high cell viability, low levels of cell aggregation, and minimal damage to cell surface proteins. High quality lymph node stromal cell preparations have been accomplished with protocols that use shorter enzyme incubations, Ca2+ and Mg2+ free media, and avoid trypsin and dispase18. However, protocols of this type have not been established for the dissociation of whole mouse skin.
Here, a protocol is described to dissociate, isolate, and enrich skin-infiltrating leukocytes from allergen-challenged mouse flank skin. Briefly, excised skin is pre-incubated in Hank&#39;s Balanced Salt Solution (HBSS) with 10% fetal bovine serum for 1 hr to soften the tissue for digestion and remove any excess dead skin or fatty tissue. This is followed by a 30 min&#160;enzymatic digestion step with 0.7 mg/mL collagenase D. Collagenase D has minimal effects on density of cell surface markers, and no effect on T cell proliferation in vitro16,18, making it highly suitable for applications involving the characterization of surface proteins. Following enzymatic digestion, discontinuous density gradient centrifugation was used to remove epithelial cells and debris from the single-cell suspension and enrich for hematopoietic cells. Importantly, this procedure avoids expensive column-based magnetic cell separation reagents and tissue dissociation machines8-11, and can be performed with equipment and materials found in a basic biomedical research laboratory. Here this protocol was used to isolate leukocytes from flank skin challenged three times with the hapten oxazolone (Ox) in previously sensitized ND4 Swiss mice (adapted from 19). Cells were analyzed using multi-parametric flow cytometry. This technique yielded a cell suspension with minimal debris and &#62;95% viability of isolated lymphocytes which were analyzed by multi-parametric flow cytometry to measure the infiltration of T lymphocytes and neutrophils into the affected skin.

<table border="0" cellpadding="0" cellspacing="0" width="1213">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">HBSS with phenol red, without calcium, without magnesium, liquid</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">55021C</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">HEPES, &#8805;99.5% (titration)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">H3375</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">EDTA disodium salt solution</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E7889</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td>
			<td style="width:179px;">MidSci</td>
			<td style="width:181px;">S01520HI</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Percoll, pH 8.5-9.5 (25&#176;C)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">P1644</td>
			<td style="width:396px;">Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Trimmer Combo Kit</td>
			<td style="width:179px;">Kent Scientific</td>
			<td style="width:181px;">CL9990-1201&#160;</td>
			<td style="width:396px;">Use the larger trimmer for shaving the flank and back.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E0753-10G</td>
			<td style="width:396px;">Dissolve in 100% EtOH at 50&#176;C for 15 minutes on a rotating plate.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Purified anti-mouse CD16/CD32 (2.4G2)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">70-0161</td>
			<td style="width:396px;">Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:457px;">anti-CD3e-BV650 (145-2C11)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">564378</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD4-APC-eFluor780 (RM4-5)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">47-0042-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD8a-BV785 (53-6.7)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">100749</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11b-eFluor450 (M1/70)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0112-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11c-eFluor450 (N418)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0114-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD44-BV711 (IM7)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">563971</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45-FITC (30-F11)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">35-0451-U025</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45R-eFluor450 (RA3-6B2)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0452-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD62L-PerCP-Cy5.5 (MEL-14)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">104431</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">108407</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">Ghost Dye-BV510</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">13-0870-T100</td>
			<td style="width:396px;">Viability dye for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">LSR Fortessa</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">N/A</td>
			<td style="width:396px;">Cell analyzer (18 parameters)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:457px;">Collagenase D from Clostridium histolyticum</td>
			<td style="width:179px;">Roche Applied Science</td>
			<td style="width:181px;">11088858001</td>
			<td style="width:396px;">Aliquot lyophilized enzyme at 5 mg/mL in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&#160; Store immediately at -20&#176;C for up to six months.</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:457px;">ND4 Swiss female mice</td>
			<td style="width:179px;">Harlan</td>
			<td style="width:181px;">0-32</td>
			<td style="width:396px;">8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#39;s IACUC guidelines.&#160;</td>
		</tr>
	</tbody>
</table>

isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation

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

Collagenase D treated splenocytes show similar levels of CD4 and CD8&#945; on T cells when compared to media-treated controls 
First, any potential effects of collagenase D on the frequency and surface abundance of lineage and activation markers on T cell subsets were assessed using secondary lymphoid tissue as a control. A suspension of splenocytes was obtained from ND4 mice and washed for 1 hr with HBSS media. Next, half the cells ...

Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

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

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

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

isolation-infiltrating-leukocytes-from-mouse-skin-using-enzymatic

Research

JoVE Journal

Immunology and Infection

21.2K Views.  Macalester College. This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols. 

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

Isolation of Brain-infiltrating Leukocytes

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

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

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

Isolation and 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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

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

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

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

Isolation of 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 ...