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>

<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. W., 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=Modern+flow+cytometry:+a+practical+approach.">Modern flow cytometry: a practical approach.</a> Clin Lab Med. 27, 453-468 (2007).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4, 648-655 (2004).</li><li>Sharrow, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+flow+cytometry.">Overview of flow cytometry.</a> Curr Protoc Immunol. Chapter 5, Unit 5.1 (2002).</li><li>Holmes, K., Lantz, L. M., Fowlkes, B. J., Schmid, I., Giorgi, 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 border="0" cellpadding="0" cellspacing="0" width="1056">
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Isoflurane</td>
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			<td style="width:140px;">1001936060</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:379px;">5- and 6-carboxyfluorescein diacetate succinimidyl ester 
			(CFSE)</td>
			<td>Invitrogen</td>
			<td>C1157</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Dimethyl sulphoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Bacille Calmette-Gu&#233;rin (BCG)</td>
			<td></td>
			<td></td>
			<td style="width:325px;">strain Pasteur, in house</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Trypan blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td style="width:325px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">EDTA</td>
			<td></td>
			<td></td>
			<td style="width:325px;">In house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PBS</td>
			<td></td>
			<td></td>
			<td style="width:325px;">In house</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Filtered water</td>
			<td></td>
			<td></td>
			<td style="width:325px;">In house</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">BD Plastipak 3 ml syringe</td>
			<td>BD Medical Surgical Systems</td>
			<td>309658</td>
			<td style="width:325px;">alternative: 5 ml syringe&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:379px;">6 well plates</td>
			<td>TPP</td>
			<td>92006</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">15 ml centrifuge tubes</td>
			<td>TPP</td>
			<td>91015</td>
			<td style="width:325px;">Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">5 ml round bottom tubes</td>
			<td>BD Falcon</td>
			<td>3272948</td>
			<td style="width:325px;">Polystyrene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Univentor&#160; 400 anaesthesia unit</td>
			<td>Univentor</td>
			<td>8323001</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Monojet 3/10ml syringe</td>
			<td>Medicarrier</td>
			<td>8881511144</td>
			<td style="width:325px;">29 G x 1/2 in.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Sterile filter unit</td>
			<td>TPP</td>
			<td>99500</td>
			<td style="width:325px;">PES membrane&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">EPPI-Pistill homogenizer</td>
			<td>Schuett biotech</td>
			<td>3.200 512</td>
			<td style="width:325px;">Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Allegra X-30R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B01145</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">B&#252;rker counting chamber</td>
			<td>VWR</td>
			<td>631-0920</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">0.5 ml screw cap microtube&#160;</td>
			<td>Sarstedt</td>
			<td>72.730</td>
			<td style="width:325px;">Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">1.5 ml Eppendorf microcentrifuge tube</td>
			<td>VWR</td>
			<td>700-5239</td>
			<td style="width:325px;">Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">70 micron cell strainers</td>
			<td>VWR</td>
			<td>734-0003</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">BD LSR-II flow cytometer, operated with FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td style="width:325px;">Discontinued. Other Flow cytometers available, minimum detection of 6 fluorochromes needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">FlowJo cell analysis software</td>
			<td>Tree Star</td>
			<td></td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Antibody</td>
			<td style="width:212px;">Clone</td>
			<td style="width:140px;">Company&#160;</td>
			<td style="width:325px;"></td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:379px;">Antibodies</td>
			<td style="width:212px;"></td>
			<td style="width:140px;"></td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">MHC-II I-A/I-E&#160;</td>
			<td style="width:212px;">M5/114.15.2</td>
			<td style="width:140px;">BD Biosciences</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD11b</td>
			<td style="width:212px;">M1/70</td>
			<td style="width:140px;">BD Biosciences</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD11c</td>
			<td style="width:212px;">HL3</td>
			<td style="width:140px;">BD Biosciences</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD16/CD32 (Fc-block)</td>
			<td style="width:212px;">2.4G2</td>
			<td style="width:140px;">BD Biosciences</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD326/EpCAM</td>
			<td style="width:212px;">G8.8</td>
			<td style="width:140px;">Biolegend</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD103</td>
			<td style="width:212px;">2E7</td>
			<td style="width:140px;">Biolegend</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD80</td>
			<td style="width:212px;">16-10A1</td>
			<td style="width:140px;">Biolegend</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">CD86</td>
			<td style="width:212px;">GL-1</td>
			<td style="width:140px;">Biolegend</td>
			<td style="width:325px;"></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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

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Immunology and Infection

12.0K 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

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

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 ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

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

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

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

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

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

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...