Name: a cfse-based assay to study the migration of murine skin dendritic cells into draining lymph nodes during infection with mycobacterium bovis bacille calmette-guérin
Uploaded: 2016-10-09T15:00:00.000+00:00
Duration: 7 min 14 s
Description: Watch this Scientific Journal Video about A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-

This work was supported by Karolinska Institutet, a project grant from the Swedish Research Council VR (Dnr 2014-2794) and a KID Doctoral Grant from Karolinska Institutet (All to A.G.R). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

NOTE: C67BL/6 mice were used for the experiments shown in this manuscript. These animals were house under specific pathogen-free conditions at the MTC animal facility, Karolinska Institutet, Stockholm, Sweden. Animal experiments were performed according to the directives and guidelines of the Swedish Board of Agriculture, the Swedish Animal Protection Agency, and Karolinska Institutet. Experiments were approved by the Stockholm North Animal Ethics Council.
1. Preparation of CFSE Stocks and Storage
<ol>
	<li>Prepare a 5 mM stock of 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in dimethyl sulfoxide. Vortex. Dispense 100 &#181;l aliquots into sterile, 0.5 ml screw cap microtubes. Store aliquots at -80 &#176;C.</li>
</ol>
2. Mice
<ol>
	<li>Use inbred male or female mice between 8 and 12 weeks of age. Sex- and age-matched animals are preferred. Make sure the necessary animal ethical permits are in place before the initiation of experiments.</li>
</ol>
3. Immobilization of Animals with Isoflurane Gas Anesthesia
<ol>
	<li>Load the anesthesia unit with isoflurane. Fill the 10 ml gas-tight glass syringe with isoflurane. Avoid introducing air bubbles. Connect the syringe and liquid inlet with the supplied tubing and move the pusher forward until the liquid in the connecting tube is visibly just about to enter the vaporizer. 
	NOTE: Do not store isoflurane in the syringe when not in use.</li>
	<li>Anesthetize animals with isoflurane in the induction chamber, with an airflow maintained at approximately 400 - 500 ml/min and the concentration of isoflurane at 3.5%. 
	NOTE: The unit will not operate without airflow.</li>
	<li>Once the animals are asleep, turn the stopcock on the anesthesia unit to route the isoflurane gas to the mask piece. Check that the mask piece is intact to avoid leakage of gas. Airflow can be maintained at 400 ml/min, alternatively decreased to 200 ml/min. Reduce isoflurane concentration to 2.6%. 
	NOTE: Anesthetic effect can be increased and/or decreased by adjusting the flow rate.</li>
</ol>
4. BCG Injections and Recovery
<ol>
	<li>Visually confirm and perform tests such as the toe pinch reflex test to ascertain that mice are immobilized/asleep on the isoflurane mask piece before performing injections.</li>
	<li>Inoculate in the hind footpad, 1 x 106 Colony-forming units of Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in 30 &#181;l of PBS using a syringe fitted with a 29 G x &#189; inch needle. 
	NOTE: The generation of mycobacterial stocks is briefly described in the original description of this procedure 3, and in substantial detail elsewhere 6. BCG is also readily available from commercial sources. The authors recommend that BCG be pulse-sonicated or passed through a syringe to disrupt clumps prior to its inoculation into mice. BCG is reiterated here as a microbial stimuli given its employment in the original description of this protocol 3 but the authors certainly encourage the testing of other microbial stimuli.</li>
	<li>Perform the same procedure described above for the injection of PBS into control mice.</li>
	<li>Monitor animals after injections to confirm that recovery from anesthesia is successful and that animals can support themselves on the injected, hind footpad.</li>
</ol>
5. CFSE Injections
<ol>
	<li>Preparation of CFSE for Injections:
	<ol>
		<li>Thaw a vial of 5 mM CFSE stock solution from -80 &#176;C. Dilute the CFSE 10-fold in PBS. Resuspend the solution thoroughly before filling the syringe in preparation for the injection.</li>
	</ol>
	</li>
	<li>CFSE Injection and Recovery:
	<ol>
		<li>Repeat the procedure for anesthetizing animals in section 3. Inject 20 &#181;l of 0.5 mM CFSE in the hind footpad which previously received BCG or PBS. 
		NOTE: The injection of CFSE should be performed the day before (24 hr) the planned date for sacrifice.</li>
	</ol>
	</li>
</ol>
6. Surgical Removal of Popliteal Lymph Node (pLN)
NOTE: Use of sterilized surgical instruments and their repeated decontamination in 70% ethanol are encouraged throughout this step.
<ol>
	<li>Euthanize the mice. Spray the animal with 70% ethanol. Pin the animal in the dorsal position, on a dissection board. 
	NOTE: The authors can recommend euthanasia through cervical dislocation.</li>
	<li>Carefully make a vertical incision in the hind thigh. Clear muscle and fat using scissors and tweezers to expose and consequently excise the pLN located deep in the fat pouch, in the hollow of the knee.</li>
	<li>Transfer the pLN to 0.5 ml sterile PBS, on ice.</li>
</ol>
7. Generation of Single-cell Suspension from pLN
<ol>
	<li>Homogenize the pLN through a 70-micron cell strainer placed in a 6 well-plate containing 5 ml PBS or fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% fetal calf serum (FCS), 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM sodium azide). Homogenize through the strainer using the back end of a 3 ml syringe. 
	NOTE: Sodium azide is toxic and should be handled with caution.</li>
	<li>Wash the strainer with another 5 ml of PBS or FACS buffer, collect the material in a 15 ml tube and pellet the cells at 277 x g (1,200 rpm, R = 172 mm) for 5 min, 4 &#176;C. Alternatively, homogenize pLNs directly in microcentrifuge tubes using polypropylene homogenizers.</li>
	<li>Based on the pellet size, resuspend the pLN suspension in an appropriate volume of PBS or FACS buffer, e.g., 200 - 1,000 &#181;l. Use a counting chamber to quantify the total cell number obtained from each LN suspension. Use trypan blue to exclude dead/dying cells.</li>
</ol>
8. Cell Staining and Flow Cytometry
<ol>
	<li>Transfer 1 - 10 x 106 cells to 5 ml round-bottom polystyrene tubes. Wash with FACS buffer, and pellet by centrifugation at 277 x g (1,200 rpm, R= 172 mm) for 5 min, 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 50 - 100 &#181;l of antibody cocktail (see Dendritic cell [DC] markers used for surface staining, Materials Table) containing 0.5 - 1 &#181;g of anti-mouse CD16/CD32 (to block unspecific Fc-mediated interactions) in FACS buffer for 30 - 45 min, on ice. Protect samples from light.</li>
	<li>After incubation, wash cells with FACS buffer and pellet by centrifugation at 277 x g (1,200 rpm, R = 172 mm) for 5 min, 4 &#176;C.</li>
	<li>Resuspend cells in 50 - 100 &#181;l of FACS buffer. Acquire data on a flow cytometer 7-10. 
	NOTE: For excellent reviews on flow cytometry we refer the reader to the following publications that relay both theoretical and practical information on this method.</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCG+Skin+Infection+Triggers+IL-1R-MyD88-Dependent+Migration+of+EpCAMlow+CD11bhigh+Skin+Dendritic+cells+to+Draining+Lymph+Node+During+CD4++T-Cell+Priming.">BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming.</a> PLoS Pathog. 11, e1005206 (2015).</li><li>Allan, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migratory+dendritic+cells+transfer+antigen+to+a+lymph+node-resident+dendritic+cell+population+for+efficient+CTL+priming.">Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.</a> Immunity. 25, 153-162 (2006).</li><li>Itano, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dendritic+cell+populations+sequentially+present+antigen+to+CD4+T+cells+and+stimulate+different+aspects+of+cell-mediated+immunity.">Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity.</a> Immunity. 19, 47-57 (2003).</li><li>Larsen, M. H., Biermann, K., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Mycobacterium+tuberculosis.">Laboratory maintenance of Mycobacterium tuberculosis.</a> Curr Protoc Microbiol. Chapter 10, Unit 10A 11 (2007).</li><li>Tung, J. 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The authors declare that they have no competing financial interests.

noDCs are specialized antigen-presenting cells that can respond to microbial challenge at body surfaces by acquiring microbial antigen and migrating to DLN via lymphatics. There, DCs present this antigen to T cells in a manner that drives primary T-cell responses. The process of DC migration from tissue to DLN is therefore highly important for understanding how a productive T-cell response is initiated. Methods to measure DC migration are consequently very useful in unfolding the above.
A mouse model was employed to study skin DC migration to DLN following infection with BCG, a live vaccine that is injected clinically in the skin. In line, BCG is injected in the hind footpad to mimic this cutaneous route of vaccination. A simple assay was developed and consequently incorporated to this model to investigate the migration of skin DC sub-populations from the BCG inoculation site in the footpad skin to the draining pLN. This protocol relies on injecting the fluorochrome CFSE into the same footpad that previously received BCG and then isolating the draining pLN 24 hr later to analyze the presence of CFSE-labeled cells by flow cytometry. Although not described in the protocol, LNs from this setup can also be prepared for analysis by confocal microscopy, to study migration processes such as the positioning of migratory cells in the LN 3. Furthermore, similar results on BCG-triggered skin DC migration have been obtained using both BCG Pasteur 1173P2 3 and BCG SSI 1331 14.
CFSE is commonly used to label cells in vitro and to track such labeled cells in vivo after cell transfers 15. In the current protocol though, it was used to directly label skin cells in situ. The molecular basis of CFSE labeling is similar to FITC, which is also an amine-reactive fluorescent dye. CFSE is however preferred over FITC for conjugation as it creates very stable carboxamide bonds 16. Moreover, CFSE is highly membrane permeable and passively diffuses into cells. Once inside, it forms intracellular amine (fluorescent) conjugates that are retained in the labeled cell 15.
The CFSE-based migration assay developed by the authors enables the identification of cells relocating from skin to DLN within a 24-hr period in response to BCG. It thus measures acute migration during a defined timeframe and allows one to investigate the ability of different, injected stimuli to trigger migration. This assay is different from the classical technique of FITC skin painting, which measures a cumulative migration event triggered by cutaneous, topical application of a contact-sensitizing agent. Thus, the CFSE-based migration assay can be employed to identify migratory skin DCs or other cells that home to the pLN after the injection of microbes, microbial products or other inflammatory stimuli in the footpad. Indeed, both neutrophils and &#947;:&#948; T cells have been shown to relocate from skin to DLN in response to BCG 17,18. In fact, the assay was recently used in a co-infection setting to show that a gut-localized nematode infection could mute BCG-triggered skin DC migration to DLN 14.
DC migration is often assessed between 24 to 72 hr in FITC skin painting since FITC-labeled DCs are difficult to detect 4 to 5 days after painting 19. Loss of the CFSE signal on labeled DCs is not an issue in the assay presented here since analysis of migration was restricted to 24 hr; the reason behind this being that the authors wanted to specifically study &#34;overnight&#34; migration events. Others interested in this assay are however encouraged to investigate earlier or even later time points for CFSE-based tracking. Moreover, since CFSE is introduced by injection it can be given at any defined time. This flexibility allowed the authors to assess DC migration between day 5 and 6 after BCG infection (Figure 2C) 3. Injecting CFSE could potentially limit the cell types accessible in situ to the labeling reaction. For instance Langerhans cells (LCs), which are DCs that reside in the top layer of the skin, the epidermis, migrate poorly in response to BCG in the CFSE-based migration assay (Figure 3) 3. However, during FITC skin painting, DCs positioned in the dermis, the layer below the epidermis, are readily labeled and migrate to the DLN faster than LCs 20,21. This suggests that cells can become fluorochrome-labeled without necessarily needing to localize to the layer of the skin where the labeling solution is applied.
As a model for BCG infection, the mouse ear provides a bona fide intradermal route of inoculation that is more in line with the clinical administration of BCG as a tuberculosis vaccine. Footpad injection on the other hand, is likely a combination of the intradermal and subcutaneous routes. That said, it requires skill and training to correctly and reproducibly deliver BCG to the dermis 22, so in clinical practice it may not always be given truly intradermal but rather subcutaneous or a combination of both. As an inoculation site the footpad does have two very clear advantages over the ear that merit mentioning. First, the immune response is concentrated to the draining pLN following a footpad injection and this facilitates the examination of responses. The authors of this report found the pLN to be the first and main LN draining the footpad 3. Others have suggested split drainage between the popliteal and subiliac LNs after injection of Evan&#39;s blue 23. Nevertheless, in regards to the ear, there is more than one ear DLN in mice and these may not be regularly present or easy to locate 24. The latter clearly introduces a liability in studies seeking to investigate responses in DLN. Second, larger volumes can be inoculated into the footpad (10 - 50 &#181;l) compared to the ear. This in turn both minimizes the liability of introducing major alterations in lymphatic flow and of having to work with very small injection volumes, which is in itself an issue when it comes to inoculating large quantities of mycobacteria. In the ear where injection volumes have to be small (1-10 &#181;l), even 5 &#181;l may alter lymphatic flow and potentially force a larger amount of the injected material directly into the DLN in an uncontrolled way. It is therefore important to account for injection volumes. This is especially important in experiments addressing the contribution of cells in ferrying bacteria to the LN. In the BCG footpad model, some BCG may have reached the DLN in the absence of cellular transport. This is based on the observation that one can still detect BCG in the DLN of mice where cell migration from the footpad was entirely ablated by local injection of pertussis toxin 3. Whether this is an indication that BCG can directly access lymphatics and reach the DLN in a truly cell-free manner remains to be determined since it cannot be excluded that mycobacteria in this case where given access to lymphatics by force of the injected volume.
A practical consideration for immunizations in the ear or footpad is that animals have to be adequately immobilized for injections to be carried out correctly and in a reproducible manner. Isoflurane gas is described in the current protocol as a method to restrain animals in preparation for footpad injections. The relatively rapid induction and recovery times for isoflurane-mediated anesthesia make it a popular method, but similar to other anesthetic agents, it affects physiology, which may interfere with experimental results 25. Indeed, both volatile and injectable anesthetics decrease lymph flow by abolishing voluntary muscle movements, reducing muscle tone, and decreasing lymphangion contraction 26. Furthermore, a 5-fold reduction in migration to DLN of DCs adoptively transferred in the footpad has been reported in anesthetized animals 27. Given the above, and since the handling procedures prior to anesthesia are likely to cause more stress to the animals than the injection itself, which is both rapid to administer and does not require a recovery step, the possibility to adequately restrain the animal without anesthetics should be considered. A mouse restrainer customized for footpad injections, where the animal can be rapidly brought to an immobilized position and inoculated in the footpad within 30 to 40 sec would be ideal to inject the mouse in the hind footpad without altering other aspects of its physiology in the process, and would be extremely useful in studies addressing cell migration through lymphatics.
In conclusion, this report highlights a novel protocol for tracking skin DCs during their mobilization to the DLN using an assay that monitors migration during a defined 24-hr window. This CFSE-based migration assay allows detection and analysis of migratory cells by flow cytometry, as detailed here, as well as by confocal microscopy 3. It provides a flexible platform for studying a variety of injected stimuli and their ability to trigger DC migration, and importantly, can be employed to track not only DCs but also other populations moving from skin to DLN during different experimental settings.

DCs localized in body surfaces sense microbes or their products, and in doing so mobilize via lymphatic vessels to the draining LN (DLN) 1,2. This relocation is needed for transport of microbial antigen to the DLN and the consequent priming of immune responses to the invading microbe. Indeed, blockade or failure of DCs to migrate from the infected tissue to the DLN mutes the T-cell response 3-5. Accordingly, assays designed to track migration at the single-cell level are useful to help ascribe migratory function to a given DC subset.
There is a large body of data on the mobilization of skin DCs to the DLN. The latter accounts for much of the knowledge on DC migration from inflamed or infected sites 2. This is not surprising since the skin houses a large population of DCs and is a highly accessible surface for experimentation in the laboratory mouse. Several techniques have thus been developed to assess the migration of murine DCs from the skin to the DLN 2. FITC skin painting is by far the most commonly used approach. In this assay the fluorochrome fluorescein-5-isothiocyanate (FITC) is prepared in a mixture with acetone and a contact irritant. This cocktail is applied to depilated or shaved skin and the accumulation of FITC-labeled cells is in turn analyzed in the DLN. Migration can also be studied by injecting the skin with fluorochrome-tagged nano- or microparticles, which enables the tracking of phagocytic cells that have internalized the fluorescent particles in the skin. Lymphatic vessels can also be cannulated to directly extract migrating DCs from lymph. This is however technically challenging, especially in mice. The number of DCs obtained for subsequent analysis is also a limiting factor.
In spite of many previous studies on skin DC migration, there remains a paucity of information regarding skin DC mobilization to DLN following vaccination with Bacille Calmette-Gu&#233;rin (BCG), the live, attenuated strain of Mycobacterium bovis used to vaccinate against M. tuberculosis. BCG is inoculated in the skin, causing a limited infection that triggers a T helper-cell type 1 response. Both the DC sub-populations that mobilize to the DLN from the BCG-infected skin and the factors that regulate their migration during this process are not fully understood. In light of the above, a simple but novel method was developed where the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) is injected in situ to track the migration of DCs into the DLN 3. C57BL/6 mice are injected in the hind footpad with an inoculum of BCG and the day before sacrifice, animals are injected in the same footpad with a high concentration of CFSE. Twenty-four hours later animals are sacrificed and the draining popliteal LN (pLN) removed and prepared for flow cytometry. This assay enables the identification of cells that migrate overnight from the footpad skin to the DLN (Figure 1).
Furthermore, gene-targeted mice can be used to help identify factors required for cells to mobilize to the DLN. Using this assay, the authors of this report have previously found that EpCAMlow CD11bhigh migratory skin DCs transport BCG to the DLN in a process regulated by Interleukin-1 receptor and the intracellular adaptor molecule MyD88 3. The protocol for this CFSE-based migration assay which originates from the above report by Bollampalli et al.3 where flow cytometry was used to detect and analyze migratory skin DCs, is herein described. The advantages and disadvantages of this assay are discussed.

<table><tbody><tr><td>Isoflurane</td><td>Baxter</td><td>1001936060</td><td></td></tr><tr><td>5- and 6-carboxyfluorescein diacetate succinimidyl ester&lt;br/&gt; (CFSE)</td><td>Invitrogen</td><td>C1157</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Bacille Calmette-Gu&amp;eacute;rin (BCG)</td><td></td><td></td><td>strain Pasteur, in house</td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>EDTA</td><td></td><td></td><td>In house</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td>In house</td></tr><tr><td>Filtered water</td><td></td><td></td><td>In house</td></tr><tr><td>BD Plastipak 3 ml syringe</td><td>BD Medical Surgical Systems</td><td>309658</td><td>alternative: 5 ml syringe&amp;nbsp;</td></tr><tr><td>6 well plates</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>TPP</td><td>91015</td><td>Polypropylene</td></tr><tr><td>5 ml round bottom tubes</td><td>BD Falcon</td><td>3272948</td><td>Polystyrene</td></tr><tr><td>Univentor 400 anesthesia unit</td><td>Univentor</td><td>8323001</td><td></td></tr><tr><td>Monojet 3/10 ml syringe</td><td>Medicarrier</td><td>8881511144</td><td>29 G x 1/2 in.</td></tr><tr><td>Sterile filter unit</td><td>TPP</td><td>99500</td><td>PES membrane&amp;nbsp;</td></tr><tr><td>EPPI-Pistill homogenizer</td><td>Schuett biotech</td><td>3.200 512</td><td>Polypropylene</td></tr><tr><td>Allegra X-30R centrifuge</td><td>Beckman Coulter</td><td>B01145</td><td></td></tr><tr><td>B&amp;uuml;rker counting chamber</td><td>VWR</td><td>631-0920</td><td></td></tr><tr><td>0.5 ml screw cap microtube&amp;nbsp;</td><td>Sarstedt</td><td>72.730</td><td>Polypropylene</td></tr><tr><td>1.5 ml Eppendorf microcentrifuge tube</td><td>VWR</td><td>700-5239</td><td>Polypropylene</td></tr><tr><td>70 micron cell strainers</td><td>VWR</td><td>734-0003</td><td></td></tr><tr><td>BD LSR-II flow cytometer, operated with FACSDiva software</td><td>BD Biosciences</td><td></td><td>Discontinued. Other Flow cytometers available, minimum detection of 6 fluorochromes needed</td></tr><tr><td>FlowJo cell analysis software</td><td>Tree Star</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td>&lt;strong&gt;Clone&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td></td></tr><tr><td>MHC-II I-A/I-E&amp;nbsp;</td><td>M5/114.15.2</td><td>BD Biosciences</td><td></td></tr><tr><td>CD11b</td><td>M1/70</td><td>BD Biosciences</td><td></td></tr><tr><td>CD11c</td><td>HL3</td><td>BD Biosciences</td><td></td></tr><tr><td>CD16/CD32 (Fc-block)</td><td>2.4G2</td><td>BD Biosciences</td><td></td></tr><tr><td>CD326/EpCAM</td><td>G8.8</td><td>Biolegend</td><td></td></tr><tr><td>CD103</td><td>2E7</td><td>Biolegend</td><td></td></tr><tr><td>CD80</td><td>16-10A1</td><td>Biolegend</td><td></td></tr><tr><td>CD86</td><td>GL-1</td><td>Biolegend</td><td></td></tr></tbody></table>

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.

The CFSE-based migration assay (Figure 1) was used together with flow cytometry to detect and characterize the CFSE-labeled cells that appear in the pLN after BCG infection in the footpad. A large portion of CFSE labeling in the pLN is found on MHC-IIhigh and CD11c+/low cells (Figure 2A), consistent with skin DCs that have migrated to skin DLN 11. Both the frequency and total number of CFSE-labeled, MHC-IIhigh CD11c+/low cells are increased in pLNs from BCG-infected mice compared to PBS-injected controls (Figure 2B-C), suggesting that BCG enhances the relocation of these cells to the DLN. Since this assay measures migration during a 24-hr period, it was possible to establish that skin DCs are still entering the DLN 5 days after infection (Figure 2C). This extends DC migration in the current model well beyond the timeline for initial activation of antigen-specific CD4+ T cells in the pLN 3. Further, migratory skin DCs express elevated levels of co-stimulatory molecules CD80 and CD86 (Figure 2D). This is consistent with previous observations from skin explant cultures and FITC skin painting 11, supporting the concept that migratory DCs are activated cells. Importantly, both LN-resident DCs (MHC-II+ CD11chigh cells) and plasmacytoid DCs (MHC-IIlow CD11clow PDCA-1+ cells), which enter inflamed LNs through high endothelial venules and not afferent lymphatics 12, where negative for CFSE (Figure 2EF), suggesting indeed that CFSE labeling occurred mainly in the footpad.
Given that MHC-IIhigh CD11c+/low cells represent not one but multiple DC sub-populations 13, the additional surface markers CD103, EpCAM (CD326) and CD11b were included in the antibody staining panel used for flow cytometric detection (Materials Table) to further characterize the population of migratory DCs (Figure 3). In doing so, a sub-population of EpCAMlow CD11bhigh cells was identified as the main migratory skin DC subset in response to BCG infection (Figure 3).
The flow cytometric data shown here was acquired on a flow cytometer equipped with 4 lasers (488, 532, 640 and 405 nm). The data was analyzed with cell analysis software. Other multi-color flow cytometers than the one specified in the Materials Table can be employed for the successful detection of the markers mentioned in the above studies, or the detection of other markers, on other cell types for that matter. For the protocol described herein, a cytometer capable of detecting 6 different fluorochromes is necessary. Importantly, the clones for each antibody used are specified (Materials Table). The choice of fluorochrome conjugate needs to be considered as this may depend on the lasers and detection channels on the flow cytometer available. Similarly, antibodies need to be titrated to determine the most appropriate dilutions for staining.
Population frequencies in BCG- and PBS-injected samples were graphed and used together with total cell counts to calculate absolute numbers of defined, gated subsets of DCs. The significance of differences in data group means was analyzed by Student&#39; s t test or ANOVA where appropriate, with a cut-off of p &#60; 0.05. Outliers were excluded from analysis following Grubbs&#39; s test for outliers.
<img alt="Figure 1" src="/files/ftp_upload/54620/54620fig1.jpg" /> 
Figure 1. Outline: BCG Footpad Infection Model and CFSE-Based Migration Assay. BCG is inoculated in the hind footpad of C57BL/6 mice. At different time points after infection, and 24 hr before sacrifice, the fluorochrome CFSE is injected in the same footpad that previously received BCG. The draining, popliteal lymph node is isolated and CFSE-labeled cells characterized by flow cytometry. <a href="https://www.jove.com/files/ftp_upload/54620/54620fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54620/54620fig2.jpg" /> 
Figure 2. Migratory Skin DCs are a Major Population Relocating to the DLN After BCG Footpad Infection. C57BL/6 mice were infected with 1 x 106 CFUs of BCG in the footpad. Twenty-four hours before sacrifice, animals were injected with 0.5 mM CFSE in the same footpad. Popliteal LNs were harvested, homogenized into single-cell suspensions and subjected to flow cytometry. For measurements made 1 day after BCG, CFSE was injected 2hr after BCG. (A and B) Zebra plots showing predominant expression of CFSE on MHCIIhigh and CD11c+/low cells in BCG-draining pLN 3 days after infection. (C) Frequency and total number of CFSE-labeled MHCIIhigh CD11c+/low cells in pLN from BCG- and PBS-injected mice were graphed at different time points. (D) Mean fluorescence intensity (MFI) for CD80 and CD86 was determined on CFSE+ and CFSEneg MHC-IIhigh CD11c+/low skin DCs in pLN 3 days after BCG infection. (E) CFSE expression within skin DCs (MHC-IIhigh CD11c+/low), LN-resident DCs (MHC-II+ CD11chigh) and (F) plasmacytoid DCs (MHC-IIlow CD11clow PDCA-1+) was determined by flow cytometry 3 days after BCG. For each experiment, at least 5 mice were used for BCG-infected groups and 3 for PBS-injected controls. Bars indicate standard error of the mean. Total number of CFSE-labeled cells within each subset are given in Bollampalli et al., PLoS Pathog 2015, 11:e1005206 3. * Denotes statistically significant differences between BCG-infected and PBS controls. One of two independent experiments shown. Figure adapted from Bollampalli et al., PLoS Pathog. 2015, 11:e1005206 3, with permission from the publisher. <a href="https://www.jove.com/files/ftp_upload/54620/54620fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54620/54620fig3.jpg" /> 
Figure 3. EpCAMlow CD11bhigh DCs are the Main Skin DC Subset Trafficking to the DLN After BCG Footpad Infection. C57BL/6 mice were injected with BCG and CFSE as in Figure 2. (A) Zebra plots showing gating strategy employed (top panel) and frequency of CFSE+ cells within each, defined subset at different time points after BCG infection (bottom panels). Frequencies within each subset may vary depending on the time point after infection. After 3 days of BCG one can expect to find approximately 0.2% MHC-IIhigh CD11c+/low cells among all LN cells. Of these, approximately 6% are CD103+ cells. For the subsets found within the MHC-IIhigh CD103negative gate, one can find approximately 42% CD11bhigh EpCAMlow cells, 27% CD11blow EpCAMlow cells and 7% LCs. (B) Total numbers of CFSE+ cells within each, defined subset at different time points after BCG infection is shown. For each experiment, at least 5 mice were used for BCG-infected groups and 3 for PBS controls. Bars indicate standard error of the mean. * Denotes statistically significant differences relative to PBS-injected controls. One of three independent experiments shown. Figure re-printed from Bollampalli et al., PLoS Pathog 2015, 11:e1005206 3, with permission from the publisher. <a href="https://www.jove.com/files/ftp_upload/54620/54620fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-

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

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 ...

a-cfse-based-assay-to-study-migration-murine-skin-dendritic-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K Views.  Karolinska Institutet. 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-Guérin.

Video: A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous ...

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

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.

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 ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin

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