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>

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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><tbody><tr><td>HBSS with phenol red, without calcium, without magnesium, liquid</td><td>Sigma Aldrich</td><td>55021C</td><td>Keep sterile until day of usage.</td></tr><tr><td>HEPES, &amp;ge;99.5% (titration)</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>EDTA disodium salt solution</td><td>Sigma Aldrich</td><td>E7889</td><td>Keep sterile until day of usage.</td></tr><tr><td>Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td><td>MidSci</td><td>S01520HI</td><td>Keep sterile until day of usage.</td></tr><tr><td>Percoll, pH 8.5-9.5 (25&amp;deg;C)</td><td>Sigma Aldrich</td><td>P1644</td><td>Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201&amp;nbsp;</td><td>Use the larger trimmer for shaving the flank and back.</td></tr><tr><td>4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td><td>Sigma Aldrich</td><td>E0753-10G</td><td>Dissolve in 100% EtOH at 50&amp;deg;C for 15 minutes on a rotating plate.</td></tr><tr><td>Purified anti-mouse CD16/CD32 (2.4G2)</td><td>Tonbo Biosciences</td><td>70-0161</td><td>Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td></tr><tr><td>anti-CD3&amp;epsilon;-BV650 (145-2C11)</td><td>Becton Dickinson</td><td>564378</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD4-APC-eFluor780 (RM4-5)</td><td>eBioscience</td><td>47-0042-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD8&amp;alpha;-BV785 (53-6.7)</td><td>BioLegend</td><td>100749</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11b-eFluor450 (M1/70)</td><td>eBioscience</td><td>48-0112-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11c-eFluor450 (N418)</td><td>eBioscience</td><td>48-0114-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD44-BV711 (IM7)</td><td>Becton Dickinson</td><td>563971</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45-FITC (30-F11)</td><td>Tonbo Biosciences</td><td>35-0451-U025</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45R-eFluor450 (RA3-6B2)</td><td>eBioscience</td><td>48-0452-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD62L-PerCP-Cy5.5 (MEL-14)</td><td>BioLegend</td><td>104431</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td><td>BioLegend</td><td>108407</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>Ghost Dye-BV510</td><td>Tonbo Biosciences</td><td>13-0870-T100</td><td>Viability dye for flow cytometry</td></tr><tr><td>LSR Fortessa</td><td>Becton Dickinson</td><td>N/A</td><td>Cell analyzer (18 parameters)</td></tr><tr><td>Collagenase D from &lt;I&gt;Clostridium histolyticum&lt;/I&gt;</td><td>Roche Applied Science</td><td>11088858001</td><td>Aliquot lyophilized enzyme at 5 mg/ml in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&amp;nbsp; Store immediately at -20&amp;deg;C for up to six months.</td></tr><tr><td>ND4 Swiss female mice</td><td>Harlan</td><td>0-32</td><td>8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#039;s IACUC guidelines.&amp;nbsp;</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 ...

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Research

JoVE Journal

Immunology and Infection

21.3K 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 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 Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Developmental Biology

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

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

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Biology

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app.jove.com/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

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

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