The authors would like to thank Dr. Marcus Bosenberg for providing YUMM 1.1 and YUMM 1.7 murine melanoma lines and Dr. Deborah J. Fowell for providing B6.Cg-Tg(CAG-tdKaede)15Utr mice in agreement with RIKEN BRC through the National Bio-Resource of the MEXT, Japan.

All animal protocols have been approved by the Institutional Animal Care and Use Committee at the Oregon Health &#38; Science University.
1. Induction of Inflammation and FITC Painting of Mouse Pinna
<ol>
	<li>In a laminar flow hood, anesthetize a C57Bl/6 mouse using vaporized isofluorane (induce at 3-5% isofluorane and maintain at 1-3% isofluorane; oxygen flow rate at 0.5-1.0 L/min). Ensure proper anesthetization by monitoring the loss of pedal reflex, involuntary movements and reduced resp...

Although the leukocyte egress from peripheral, non-lymphoid tissues is critical for the initiation and resolution of immune responses, the molecular mechanisms that govern egress are poorly understood. This gap in knowledge is largely due to ready availability of tools for the quantification in vivo. Here, we describe the use of photoconvertible mice (Kaede-Tg) to quantify endogenous leukocyte egress from the skin and tumors and provide a direct head-to-head comparison with FITC paint in inflammatory and infecti...

Peripheral tissue immune responses are shaped not only by leukocyte recruitment to the sites of inflammation but also by mechanisms that regulate their subsequent retention. Thus, protective immunity is dictated by cumulative cellular and molecular mechanisms that determine whether a leukocyte enters, stays within, or rather migrates out of peripheral tissue via lymphatic vessels. Importantly, the propensity for leukocytes to exit tissue through lymphatic vessels (termed egress) is linked to their specialized functions. Dendritic cells (DC) acquire migratory behavior in response to maturation signals leading to antigen transport and presentation in draining lymph nodes (dLN), a process that is necessary for adaptive immunity1. Scavenging myeloid cells, such as macrophages and neutrophils, serve to clear apoptotic debris through phagocytosis. During bacterial infection, neutrophils egress tissue and ultimately undergo apoptosis in dLNs2 and in a model of DSS-induced colitis, data supports the hypothesis that macrophage egress is necessary to resolve local inflammation3. Whether neutrophil and macrophage egress occurs in all inflammatory contexts, however, is unknown. Evidence for T lymphocyte egress from steady state4,5,6,7, infected8, and inflamed4,9,10,11,12 peripheral, non-lymphoid tissues indicates that T cells actively recirculate, though the tissue-based signals that drive this exit remain poorly understood. Several studies have identified signals necessary for directional migration towards draining lymphatic capillaries and subsequent egress including chemokine (C-C motif) ligand 21 (CCL21) and its receptor CCR74,11,13, chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR42,14, and sphingosine-1-phosphate (S1P)10,15,16. These mechanisms are not active in all contexts, however, and whether they determine egress of all cell types remains an open question. Importantly, further insight into the mechanisms that govern egress and its functional relevance in disease requires quantitative in vivo methods of analysis.
Several methods have been used to quantify egress in multiple animal models in vivo including direct cannulation of lymphatic vessels, adoptive transfer of ex vivo labeled leukocytes, transdermal application of fluorescent tracers, injection of labeled particles, and in vivo photoconversion17,18. Direct cannulation of afferent mouse lymphatic vessels is difficult and limited in small animals by the volumes of fluid that can be collected. Thus, cannulation has largely been performed in large animals (e.g., sheep) where such surgical manipulations are practical. These studies provide direct evidence for the presence of both lymphoid and myeloid cells in lymph10,19,20. Furthermore, ovine models reveal that acute and chronic inflammation increased lymphocyte presence in lymph by nearly 100-fold10,21.
Adoptive transfer of labeled and genetically manipulated lymphocytes has importantly revealed that CCR7 is required for the egress of CD4+ T cells from acutely inflamed skin5,11, while the pretreatment of lymphocytes with the small molecule S1P receptor agonist, FTY720, only partially inhibits their egress10. Interestingly, the egress of transferred lymphocytes from chronically inflamed skin is CCR7-independent10, but may partially require CXCR49. Adoptive transfer experiments, however, deliver non-physiological numbers of ex vivo activated and labeled lymphocytes into tissue through injection, which alters the biomechanical environment of tissues and elevated interstitial fluid pressures that open initial lymphatic capillaries and alter their transport properties22. As an alternative, transdermal application of fluorescein isothiocyanate (FITC) in the presence or absence of dermal irritants (e.g., dibutyl phthalate, DBP) or infection23,24 allows for the tracking of phagocytic cells that accumulate tracer and migrate to dLNs. Similarly, fluorescently labeled tumors provide a means to track phagocytic cells that have engulfed tumor material25. These methods have provided important insight into the mechanisms that govern DC egress13,14,17,26,27 but are unable to track non-phagocytic lymphocytes and, interpretation can be complicated by free lymphatic drainage of soluble FITC thus labeling non-migratory, LN resident DCs.
Alternatively, intravital microscopy is a powerful tool that allows for in vivo tracking of physiologically relevant leukocyte populations in real time28,29. Used in combination with the reporter mice and antibody-based in vivo immunofluorescent labeling, intravital microscopy has revealed the complex spatial and temporal dynamics of immune cell trafficking, including interstitial migration30, transmigration across the lymphatic endothelium, passage within the lymphatic lumen, and migration upon LN entry28,31. Broad adoption of intravital imaging techniques is limited by expense, necessary expertise for set up, and limited throughput for quantifying multiple cell types. Still, coupling quantitative methods that analyze population dynamics tissues with intravital imaging will provide additional and important mechanistic insight with respect to the mechanisms of motility and migration toward and within lymphatic capillaries18,31,32.
Consequently, in vivo photoconversion has emerged as a method that allows for in situ labeling, independent of phagocytic activity, and for the quantification of physiological leukocyte egress (when coupled with flow cytometry) in the absence or presence of challenge. Kaede-Tg mice constitutively express a protein isolated from stony coral that exhibits green fluorescence (Kaede green) until exposed to violet light, after which it irreversibly converts to red fluorescence (Kaede red)33. Photoconverted cells can be tracked as they egress from peripheral tissue sites and accumulate in dLNs. This and other similar photoconvertible mouse models34,35 have revealed important biology including constitutive egress of regulatory T cells from skin36, CXCR4-dependent B cell egress from Peyer&#39;s patches37, mobilization of resident memory T cells upon peptide re-challenge38, and broad leukocyte egress from tumor microenvironments39. Herein, we perform a head-to-head comparison of photoconversion with transdermal FITC application in the context of cutaneous inflammation and infection to allow for direct comparison of existing data with the photoconvertible method. Furthermore, we demonstrate photoconversion in implanted tumors and describe the conversion efficiency and selective egress from tumor microenvironments. As such, we argue that further application of these methods is needed to elucidate the critical biology of leukocyte egress from tumors, which will have significant implications for interpreting intratumoral leukocyte complexity, anti-tumor immunity, and response to therapy.

<table>
	<tbody>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>4536282001</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver-LED-405B light source with optical fiber and collimtor</td>
			<td>Prizmatix Ltd</td>
			<td>V8144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I</td>
			<td>Sigma-Aldrich</td>
			<td>F4274</td>
			<td></td>
		</tr>
		<tr>
			<td>dibutyl phthalate</td>
			<td>Sigma-Aldrich</td>
			<td>524980</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Macron Fine Chemicals</td>
			<td>2440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>29-guage syringes</td>
			<td>Exel International</td>
			<td>26029</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans Blue</td>
			<td>Sigma-Aldrich</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um cell strainers</td>
			<td>VWR</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Caisson</td>
			<td>HBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Anti-mouse CD16/CD32</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-M001</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605 CD11c (clone N418)</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 MHCII (clone M5/114.15.2)</td>
			<td>BD Pharmingen</td>
			<td>562363</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421 CD3e (clone 145-2C11)</td>
			<td>Biolegend</td>
			<td>100341</td>
			<td></td>
		</tr>
		<tr>
			<td>APC CD8a (clone 53-6.7)</td>
			<td>TonBo Biosciences</td>
			<td>20-0081-u100</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7 CD45 (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103116</td>
			<td></td>
		</tr>
		<tr>
			<td>BV650 CD19 (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115541</td>
			<td></td>
		</tr>
		<tr>
			<td>PercCP-Cy5.5 Ly6C (clone HK1.4)</td>
			<td>Biolegend</td>
			<td>128011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 F4/80 (clone BM8)</td>
			<td>Biolegend</td>
			<td>123121</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7 Ly6G (clone 1A8)</td>
			<td>Biolegend</td>
			<td>127623</td>
			<td></td>
		</tr>
		<tr>
			<td>BV711 CD11b (clone M1/70)</td>
			<td>Biolegend</td>
			<td>101241</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605 CD45 (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103155</td>
			<td></td>
		</tr>
		<tr>
			<td>BV711 CD4 (clone RM4-5)</td>
			<td>BD Biosciences</td>
			<td>563726</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (Fraction V)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anit-Rat and Anti-Hamster Igk / Negative Control Compensation Particle Set</td>
			<td>BD Biosciences</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10 Software</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying leukocyte egress via lymphatic vessels from murine skin and tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

We first sought to replicate photoconversion results published in the literature to evaluate the efficiency and determine the associated inflammation in the mouse skin. The ear pinna was exposed to 100 mW violet light (405 nm) for 3 min as previously described33. Single cell suspensions generated from the ear skin or cervical dLNs immediately following the exposure revealed a 78% conversion efficiency of all CD45+ leukocytes in the skin with no converted...

Watch this Scientific Journal Video about Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors at JoVE.com

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Photoconversion provides a trackable in vivo method for the quantitative analysis of leukocyte egress for a mechanistic insight into leukocyte residence and exit dynamics from multiple tissue sites in diseased states. Well, here we demonstrate the utility of leukocyte tracking from cutaneous tumors. The application of this assay to other tissues and diseases is limited only by the ability to administer light.The main advantage of this technique is that to allows the quantification of endogenous, rather than transferred immune cell populations as a egress from inflamed tissues in vivo. To induce inflammation, after confirming a lack of response to toe pinch, use a micropipette to administer 20 microliters of dibutyl phthalate or DBP, to the ventral side of the ear pinna of an anesthetized Kaede transgenic mouse. After allowing the DBP to dry, pull the ear through a slit in a piece of aluminum foil and use double-sided tape to secure the ear flatly to the foil with the dorsal side facing upward.Then, position that you're directly under a 405 nanometer light source and photoconvert for three minutes at 100 milliwatts of power. For vaccinia infection, use a micropipette to administer 5 times 10 to the 6th plaque forming units of vaccinia virus in 10 microliters of PBS on to the ear pinna of an anesthetized Kaede transgenic mouse and poke the pinna 25 times. 24 hours later, photoconvert the pinna as just demonstrated.At the appropriate experimental time point, harvest the pinnae and the cervical and inguinal lymph nodes and use two pairs of tweezers to peel apart the ventral and dorsal side of each pinna. Then place the lymph nodes and the pinna pieces, with the inside of the ear facing down, into individual wells of a 24-well plate containing 1 milligram per milliliter of Collagenase D and 80 units per milliliter of Dnase diluted in HBSS containing calcium and magnesium. Use two 29 gauge needles to tease open each lymph node capsule and place the plate at 37 degrees Celsius for 30 minutes.At the end of the incubation, press the digested pinna and lymph node tissues through separate 70 Micron nylon cell strainers to obtain single cell suspensions of each tissue. When the tumors have grown to the appropriate experimental size, shave any newly regrown fur around the tumor site and pull the tumor through a circular hole cut in a piece of aluminum foil. Position the tumor directly below the 405 nanometer light source and photoconvert for 5 minutes at 200 milliwatts of power.24 hours after the photoconversion, harvest the tumors and brachial and inguinal lymph nodes from each animal and use scissors to mince the tumors within individual wells of a 24-well plate containing Collagenase D and DNase in HBSS. Place the lymph nodes into separate wells and tease open the capsules as just demonstrated. After incubating the tumors for one hour and the lymph nodes for a half hour at 37 degrees Celsius, press the digested tissues through separate 70 Micron strainers to obtain single cell suspensions of each tissue.To analyze the single cell suspensions by flow cytometry, first collect the spleen or blood from a Kaede transgenic mouse and lyse the red blood cells with ammonium chloride potassium buffer. Split the cells into one unconverted Kaede FITC and one converted Kaede PE population and resuspend the single color Kaede PE cells in one milliliter of PBS in one well of a 24-well plate, then photoconvert the cells for 5 minutes under a 405 nanometer light source at 100 milliwatts of power. Next use the photoconverted Kaede PE cells and the unconverted Kaede FITC cells to set the photomultiplier voltages to 70 to 80%of the total range of each fluorescent channel.Once the voltages for the Kaede proteins have been set, compensate for all of the other primary antibody stains using single fluorophore labeled compensation beads, according to the manufacturer's instructions. Then, run the samples on the flow cytometer according to standard flow cytometric analysis protocols. Single cell suspensions generated from the ear skin or cervical draining lymph nodes immediately following photoconversion exposure reveal a 78%conversion efficiency of all CD45 positive leukocytes in the skin, with no converted cells observed in the draining lymph nodes.Quantification of the numbers of infiltrating CD45 positive leukocytes in photoconverted ears zero and 24 hours post-conversion, reveals that violet light exposure causes a statistically significant increase in leukocyte infiltration. This increase in CD45 positive leukocyte infiltration however, is small relative to the infiltration induced by a potent inflammatory stimulus such as vaccinia virus. Photoconversion also increases vascular permeability in the ear pinnae as measured by Miles assay, together indicating that effects of violet light on tissue inflammation should be considered when optimizing dose, exposure time and data interpretation.Flow cytometric analysis of dissociated tumor cells from photoconverted animals reveals a significant conversion of intratumoral CD45 positive cells from Kaede FITC positive to Kaede PE positive, while draining lymph node cells remain unconverted. The photoconversion efficiency of CD45 positive leukocytes varied significantly between tumor types and sizes, with CD45 positive leukocytes within small melanoma tumors demonstrating the most efficient photoconversion. As the tumor volumes increase, the conversion efficiency decreases to around 50%Notably, melanin has a striking impact on photoconversion efficiency with a maximum photoconversion of CD45 positive cells observed within melanin-producing tumors of only about 30%in smaller tumors, that drops to 10%as the tumor volumes approach 400 cubic millimeters.The examination of tumor draining lymph nodes 24 hours after photoconversion reveals the presence of Kaede red positive immune cells of various phenotypes, thus demonstrating leukocyte emigration from the photoconverted tumor. After its development, this technique paved the way for researchers in the field of Immunology to uncover the kinetics of steady state and inflamed leukocyte turnover in peripheral tissue. While attempting this procedure, it is important to ensure that only the tissue of interest is photoconverted, as inadvertent photoconversion of the surrounding tissue will compromise your results.Individuals new to this method should optimize the exposure time for conversion of their tissue, as well as duration of the times before the tissue collection occurs based on the dynamics of leukocyte of interest.

quantifying-leukocyte-egress-via-lymphatic-vessels-from-murine-skin

Research

JoVE Journal

Biology

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...