Name: isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation
Uploaded: 2016-01-25T18:00:00
Description: Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

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
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	<li>Day 0
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		<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;un...

<ol>
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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 ...

The overall goal of this procedure is to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient separation. This method can be applied to the investigation of cellular and molecular mechanisms in many mouse models of skin pathologies, including atopic dermatitis. Though this method can provide insight into skin-infiltrating leukocyte biology, it can also be applied to the investigation of other resident cell populations.Generally, individuals new to this method will struggle with the skin harvesting, which includes a complete removal of the excess adipose and connective tissues. Visual demonstration of this method is critical, as skin harvesting and density gradient handling are difficult to learn, and require precision and practice. At the appropriate time point after treatment, use 10 centimeter surgical scissors to carefully excise a 25 by 25 millimeter area of shaved flank skin from an adult eight to twelve-week-old female ND4 Swiss-Webster mouse.Use a clean, sharp surgical blade to scrape away the excess fat and connective tissue from the skin, then transfer the skin into a 50 milliliter conical tube containing 10 milliliters of room temperature HBSS supplemented with EDTA, HEPES, and FBS, and predigest the tissue on a rotating plate for 30 minutes in a dry, non-gassed, 37 degree Celsius incubator. At the end of the incubation, vortex the tube vigorously for ten seconds and filter the tissue slurry through a 70 micron strainer. Next, use a pair of forceps to transfer the flank skin pieces from the strainer into a new 50 milliliter tube containing ten milliliters of fresh, room temperature HBSS media, supplemented with EDTA, HEPES, and FBS, then use a 12.5 centimeter pair of surgical dissecting scissors to mince each piece of flank skin so that the average piece of tissue is approximately 2.2 by 2.2 millimeters in size.When all of the pieces have been minced, return the tube to the rotating plate for another 30 minute incubation. Vortex the tube vigorously for 10 seconds at the end of the incubation and filter the contents through the 70 micron strainer. Collect the remaining flank skin pieces in a new 50 milliliter tube containing 10 milliliters of HBSS supplemented with 0.7 milligrams per milliliter of Collagenase D, and return the tube to the rotating plate for another 30 minute incubation.After another 10 seconds of vortexing, filter the contents of the tube again, this time discarding the debris on the strainer, then fill the tube with up to 50 milliliters of HBSS, EDTA, HEPES, FBS, spin down the cells and decant the supernatant. Add three milliliters of room temperature, 67 percent density gradient centrifugation medium in PBS to a new 15 milliliter conical tube, and resuspend the cell pellet in five milliliters of room temperature 44 percent density gradient centrifugation medium in HBSS with phenol red. Setting the pipettor to the lowest speed, place the pipette on the side of the tube and gently layer the cells on top of the 67 percent density gradient centrifugation medium.When working with density gradients, take care to avoid making bubbles and to minimize any disruptions to the gradient interface. Then separate the cells, using a five milliliter transfer pipette to collect the cells at the interface at the end of the centrifugation. As gently layering the gradient and carefully extracting the interface of the gradients are critical steps, it is wise to practice the density gradient centrifugation medium layering and interface removal with the suspension of murine splenocytes before attempting the procedure with the experimental cells of interest.Finally, wash the collected cells in ice-cold PBS supplemented with FBS in a 15 milliliter conical tube and count the number of viable cells by trypan blue exclusion. CD4 and CD8-alpha surface expression are not affected by Collagenase D digestion, as the same number of CD4 positive and CD8-alpha positive cells is detected with or without enzymatic digestion. Further, Collagenase D digestion exhibits no effect on the surface density of the T cell activation markers CD44 and CD62L on CD4 positive T cells.After gradient separation, about 250 viable immune cells per square millimeter of flank tissue are isolated from Oxazolone, or Ox-challenged mice, versus only four viable immune cells per square millimeter of tissue in vehicle-challenged mice, previously sensitized with Ox.95 percent of the low forward and side scatter single cells in Ox-challenged mice, and 71 percent of these cells in vehicle-challenged controls are live CD45 positive cells, equivalent to an approximately 67 fold increase in the infiltrating CD45 positive immune cell population in the flank skin following three daily Ox challenges. Indeed, Ox challenge results in the pronounced infiltration of T cells and neutrophils into the flank skin, with 42 percent of the single, live CD45 positive cells in Ox-treated animals, apparent Gr-1 positive neutrophils, and approximately 84 percent CD3 positive T cells expressing CD8-alpha, or CD4, compared to control, ethanol-treated animals. Once mastered, this technique can be completed in about three hours if it's performed properly.While attempting this procedure, it's important to remember to handle the density gradient carefully. Don't forget to always use room temperature density gradient centrifugation medium when performing this procedure. Following this procedure, other methods like flow cytometric analysis, cell sorting, cell transfer, or in vitro cell culture can be performed to evaluate the phenotype and function of the skin-infiltrating leukocytes.After watching this video, you should have a good understanding of how to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient centrifugation.

Research

JoVE Journal

Immunology and Infection

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