Name: quantifying leukocyte egress via lymphatic vessels from murine skin and tumors
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors at JoVE.com

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 respiratory rate.</li>
	<li>Lay an ear flat with the ventral side of the ear facing upwards. Pipet 20 &#181;L of 5% FITC solution dissolved in 1:1 acetone:dibutyl phthalate (DBP) to the ventral side of ear pinna. Allow the ear to dry for a few seconds.</li>
	<li>24 h after FITC application, euthanize the mice via carbon dioxide exposure followed by cervical dislocation. Collect the ear pinnae into PBS by separating the ears from the head using scissors. Collect the cervical dLNs and inguinal non-draining LNs into PBS using tweezers to separate the LNs from surrounding tissues. Inguinal non-draining LNs will serve as negative controls for the presence of FITC+/Kaede red+ leukocytes. Dispose of carcasses per institutional protocol. 
	Note: The optimal time post photoconversion for analysis will depend on the cell type and known migratory behaviors. Dendritic cells, for example, can be detected in dLNs as early as 6 h post DBP application.</li>
</ol>
2. Induction of Inflammation and Photoconversion of Mouse Pinna
<ol>
	<li>In a laminar flow hood, anesthetize a Kaede-Tg mouse (background C57Bl/6) by intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine dissolved in saline. Ensure proper anesthetization as described in Step 1.1.</li>
	<li>Lay an ear flat with the ventral side of the ear facing upwards. Pipet 20 &#181;L of a 1:1 acetone:DBP to the ventral side of the ear pinna. Allow the ear to dry for a few seconds.</li>
	<li>After DBP application, cut a slit in a piece of aluminum foil and pull the ear through the slit to expose the ear to the violet light source. Lay the ear flat with the dorsal side facing upward using double-sided tape to secure the ear to the foil.</li>
	<li>Position the ear directly under the light source, and photoconvert for 3 min using a 405 nm light source at 100 mW power.</li>
	<li>24 h after photoconversion, euthanize the Kaede-Tg mice and harvest the ear pinnae, cervical LNs, and inguinal LNs as described in Step 1.3. 
	Note: In addition to considerations cited in Step 1.3, the rates of proliferation will determine the timing of analysis as the loss of Kaede red will occur in rapidly dividing cells.</li>
</ol>
3. Vaccinia Infection of Mouse Pinna and FITC Application
<ol>
	<li>Anesthetize a C57Bl/6 mouse with vaporized isofluorane as described in Step 1.1.</li>
	<li>Lay the ventral side of the ear flat. Pipet 5 x&#160;106 plaque-forming units (PFU) of vaccinia virus (VacV) diluted in 10 &#181;L of PBS onto the ear pinna. Using a 29-gauge needle, poke the pinna 25 times40.</li>
	<li>24 h post-infection, anesthetize VacV-infected mice with isofluorane as described in Step 1.1 and pipet 20 &#181;L 5% FITC dissolved in acetone onto the ventral side of the ear pinna. Allow the ear to dry for a few seconds.</li>
	<li>24 h after FITC application, euthanize the mice and collect the ear pinnae, cervical LNs and inguinal LNs as described in Step 1.3. 
	Note: FITC may be applied at any time point post infection to determine DC trafficking at various 24 h intervals. We previously reported that DC migration to dLNs is maintained at similar levels from day 1 to 3 post infection41.</li>
</ol>
4. Vaccinia Infection of Mouse Pinna and Photoconversion.
<ol>
	<li>Anesthetize a Kaede-Tg mouse with vaporized isofluorane as described in Step 1.1.</li>
	<li>Infect the ear pinna with VacV as described in Step 3.2.</li>
	<li>24 h post-infection, anesthetize VacV-infected Kaede mice as described in Step 2.1 and perform photoconversion as described in Steps 2.3-2.4.</li>
	<li>24 h after photoconversion, euthanize the mice and harvest the ear pinnae, cervical LNs, and inguinal LNs as described in Step 1.3. 
	Note: As mentioned above in Step 3.4, photoconversion can be administered at any time point post infection.</li>
</ol>
5. Ear and Lymph Node Processing for Flow Cytometry
<ol>
	<li>Create single cell suspensions of ear pinnae: peel apart the ventral and dorsal sides of the ear pinna using two pairs of tweezers and place with the inside of the ear facing down into the wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in Hank&#8217;s Buffered Saline Solution (HBSS) (containing Ca2+ and Mg2+). Incubate at 37 &#176;C for 30 min. Press the digested tissue through a 70 &#181;m nylon cell strainer.</li>
	<li>Create single cell suspensions from LNs: place LNs in wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in HBSS. Tease open the lymph node capsule using two 29-gauge needles, and then incubate the lymph nodes at 37 &#176;C for 30 min. Press the digested tissue through a 70 &#181;m nylon cell strainer.</li>
</ol>
6. Intradermal Melanoma Tumor Implantation and Photoconversion.
<ol>
	<li>Shave the fur from the center of the back of a Kaede-Tg mouse using an electric razor.</li>
	<li>Position a 29-gauge needle in the center of the back between the left and right upper scapulae, and intradermally inject 5 x&#160;105 tumor cells (diluted in 50 &#181;L of saline) into the skin of Kaede-Tg mice. Tumors must be carefully positioned to ensure lymphatic drainage to specified lymph nodes (i.e., left and right brachial LNs for tumors placed in the middle of the upper back). Avoid placing the tumor above dLN as this can result in direct photoconversion of the LNs through the skin/tumor.</li>
	<li>Allow the tumors to grow to desired size (100-650 mm3).</li>
	<li>One day prior to tissue collection, anesthetize a Kaede-Tg mouse as described in 2.1 and shave any newly regrown fur around the tumor.</li>
	<li>Cut a circular hole in aluminum foil and pull the tumor through to expose the tumor to the light source. Cut the hole slightly smaller than the tumor to prevent the tumor from falling back through the hole and minimize the conversion of adjacent, non-tumor skin.</li>
	<li>Position the tumor directly below the light source and photoconvert for 5 min using a 405 nm light source at 200 mW power.</li>
	<li>24 h after photoconversion, euthanize the mice as described in Step 1.3. Collect the tumors, brachial dLNs, and inguinal non-draining LNs into PBS. Cut the tumors away from the surrounding skin using scissors and remove LNs as described in Step 1.3.</li>
</ol>
7. Tumor and Lymph Node Processing for Flow Cytometry
<ol>
	<li>Create single cell suspensions from the tumors: mince the tumors with scissors into wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in HBSS. Incubate at 37 &#176;C for 1 h. Press the digested tissue through a 70 &#181;m nylon strainer.</li>
	<li>Create single cell suspension of lymph nodes as described in Step 5.2.</li>
</ol>
8. Antibody Staining for Flow Cytometry
<ol>
	<li>After digesting the tissues into single cell suspensions, perform standard flow cytometry staining techniques to label the cells with markers of interest. Briefly, pipet 2 x 106 cells into a 96-well plate. Incubate the samples with Fc block (1 &#181;g/mL) for 20 min on ice; wash twice with FACs buffer (1% bovine serum albumin in PBS). Add live/dead stain (diluted in PBS) to the samples and incubate for 15 min on ice; wash twice with FACs buffer. Incubate with primary antibodies (Table of Materials) diluted in FACs buffer for 30 min on ice; wash twice with FACs buffer. 
	Note: Kaede green and Kaede red fluorescence overlaps with FITC and PE fluorophores; thus, FITC and PE-conjugated antibodies cannot be used in combination with Kaede proteins.</li>
	<li>After staining, run the samples on a flow cytometer. Alternatively, fix the cells with 2% PFA.</li>
</ol>
9. Flow Cytometry Analysis
<ol>
	<li>Set the FITC (Kaede green) and PE (Kaede red) channel PMT voltages to 70-80% of the total range (center population at approximately 104) using ex vivo Kaede green or Kaede red single-color splenocytes or blood. 
	Note: Do not compensate for the Kaede green (FITC) and Kaede red (PE) channels as this will result in greater signal spread.
	<ol>
		<li>To create single-color Kaede controls, collect a spleen or blood from a Kaede-Tg mouse. Lyse the red blood cells with ACK buffer, then divide the cells into two groups: unconverted Kaede green and converted Kaede red.</li>
		<li>For single color Kaede red controls, suspend the cells in 1 mL of PBS and photoconvert in a 24-well plate for 5 min using a 405 nm light sources at a power of 100 mW. Use these cells to set the Kaede green (FITC) and Kaede red PMT voltages on the flow cytometer (Step 9.1).</li>
	</ol>
	</li>
	<li>Once the voltages for the Kaede proteins has been set, compensate for all other primary antibody stains using single-fluorophore labeled compensation beads per manufacturer&#8217;s instructions.</li>
	<li>Run the samples on a flow cytometer to collect data. 
	Note: The Kaede protein is continually being produced by the cells. This means that 24 h after photoconversion, converted cells will be double positive for Kaede green and Kaede red as newly synthesized Kaede (green) protein has accumulated in the cell.</li>
	<li>Analyze the data using FlowJo software or a similar software.</li>
</ol>

The authors have no conflicts to disclose.

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 ...

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

Research

JoVE Journal

Biology

8.5K Views.  Oregon Health and Science University. Fotoconversie biedt een traceerbare in vivo methode voor de kwantitatieve analyse van leukocyte uitgang voor een mechanistisch inzicht in leukocytenverblijf en exitdynamiek van meerdere weefsellocaties in zieke toestanden. Nou, hier tonen we het nut van leukocyten tracking van cutane tumoren. De toepassing van deze test op andere weefsels en ziekten wordt alleen beperkt door de mogelijkheid om licht toe te dienen.Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt de...

Video: Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Cancer Research

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Immunology and Infection

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.

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 ...

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 ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Medicine

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

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 ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

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