The authors thank Patricia Baldacci, Vanessa Lagal and Sabine Thiberge for critical reading, Irina Dobrescu and Sabine Thiberge for help in taking pictures and Pauline Formaglio for teaching in vivo imaging of sporozoite motility in the mouse skin. We also would like to thank Marek Szatanik and the Center for Production and Infection of Anopheles (CEPIA-Institut Pasteur) for mosquito rearing. This study was supported by the AXA Research Fund&#160;and funds from the Laboratoire d&#8217;Excellence &#8220;Integrative Biology of Emerging Infectious Diseases&#8221; (grant no. ANR-10-LABX-62-IBEID).

All procedures were approved by the committee of the Pasteur Institute and by the local Ethics Committee on Animal Experimentation (Ethical committee IDF-Paris 1, Paris, France; agreement number: 2012-0015) and performed in accordance with the applicable guidelines and regulations.&#160;
1. Materials and Reagents
<ol>
	<li>Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously21.</li>
	<li>Use parasites Plasmodium berghei ANKA clone expressing the Green Fluorescent Protein (GFP) gene under the control of the constitutive Heat Shock Protein 70 (HSP70) promoter. This yields bright fluorescence throughout the parasite life cycle22.</li>
	<li>Use female C57BL/6JRj mice (7 week-old). 
	NOTE: All the reagents used in the described protocol are listed in the Table of Materials/Equipment.</li>
</ol>
2. Radiation-attenuated Sporozoite Isolation from Mosquito Salivary Glands
<ol>
	<li>Between 18-25 days after the infectious blood meal, collect and cold-anesthetize mosquitoes into a 15 ml tube on ice as described previously21. Carefully transfer them onto a Petri dish by inverting the tube. Maintain the insects on ice and expose the mosquitoes to a 12 krad dose of &#947;- or x-irradiation.</li>
	<li>Isolate attenuated sporozoites from irradiated mosquito salivary glands as previously described21. Collect infected salivary glands in a small volume (around 15 &#956;l) of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) on ice and gently crush them to release sporozoites. 
	NOTE: The injection of a highly concentrated parasite suspension in a small volume being critical, it is therefore essential to harvest a sufficient number of salivary glands from a high number of preselected well-infected mosquitoes, as evidenced by their robust expression of green-fluorescence.</li>
	<li>Filter the sporozoite suspension through a 35 &#181;m cell strainer cap adapted to a low adhesion 1.5 ml microcentrifuge tube in order to eliminate clumps and mosquito debris.</li>
	<li>Count the total number of isolated sporozoites as previously described21 and adjust the concentration of parasite suspension with cold 1x DPBS to obtain 83,000 sporozoites/&#956;l. Store the parasites on ice while continuing the next steps.</li>
	<li>Collect the equivalent number of salivary glands from uninfected mosquitoes that will be used as negative control and process the sample as described in steps 2.2 and 2.3. Dilute the sample as indicated above to obtain similar concentration of salivary gland extracts in the suspension. 
	NOTE: Sporozoites can be stored on ice for a maximum of 2 hr. However, ideally they are injected within an hour after crushing the salivary glands.</li>
</ol>
3. Injection of Sporozoites into the Dermal Layer of the Ear
<ol>
	<li>Anesthetize animals by intraperitoneal injection of ketamine (50 mg/kg)/xylazine (5 mg/kg) mixture. Wait 5-8 min for the mouse to be in a state of unconsciousness and apply ophthalmic ointment to the eyes to prevent corneal drying. Evaluate the depth of anaesthesia by performing a gentle toe pinch on both rear feet. 
	NOTE: The dose of anaesthesia lasts for less than 20 min, so the next steps must be done quickly.</li>
	<li>Stabilize the ear pinna of the mouse by fixing the ventral side with clear tape previously placed under a stereomicroscope (Figure 1A). Gently press the ear to avoid damage of the vasculature.</li>
	<li>Load a 10 &#956;l syringe/35 gauge bevelled needle with 0.6 &#181;l of resuspended parasites10.</li>
	<li>Deliver the sporozoite suspension in 4 injection sites (0.15 &#181;l per site) by carefully inserting the needle on the dorsal side of the ear (Figure 1A), beneath the epidermis, with the bevel up, taking care to avoid any blood vessels. Allow the needle to remain in the dermis for few sec to prevent backflow of the injectant before removing it. Observe a characteristic papule at each injection site at the end of the procedure (Figure 1B and 1C).</li>
	<li>After injection, carefully remove the ear from the tape.</li>
	<li>Inject the contralateral ear with the same dose of parasites by following the steps 3.2-3.4.</li>
	<li>Inject 2 additional mice with either 0.6 &#181;l of 1x DPBS or 0.6 &#181;l of 1x DPBS + salivary gland extract from uninfected mosquitoes in order to evaluate the inflammatory response mediated by the injection alone and the deposition of mosquito material in the dermis.</li>
	<li>&#160;Monitor mouse recovery from anaesthesia before placing it back into the cage. 
	NOTE: The proper sporozoite deposition in the skin can be monitored by fluorescence microscopy (Figure 2A and 2B), the mouse being prepared as described previsously23.</li>
</ol>
4. Skin and Draining Lymph Node Dissection&#160;
<ol>
	<li>Prepare standard medium (SM) as follows: Dulbecco&#39;s Modified Eagle Medium (DMEM) + 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplemented with 400 U/ml collagenase and 50 &#956;g/ml deoxyribonuclease (DNase).</li>
	<li>Prepare two 6 well flat-bottom tissue culture plates on ice with 4.5 ml SM + 70 &#956;m cell strainer per well for dLN samples (Plate A) and 4.5 ml SM per well for skin samples (Plate B).</li>
	<li>At the desired time point following sporozoite inoculation, deeply anesthetize the mouse by intraperitoneal injection of ketamine (125 mg/kg)/xylazine (12.5 mg/kg) mixture prior cervical dislocation. Make sure the mouse demonstrates signs of clinical death and start the dissection immediately. 
	NOTE: The kinetics of myeloid cell recruitment can be examined at different times after injection (i.e. 2 hr, 4 hr and 24 hr as previously described14).</li>
	<li>Disinfect the inoculated ear and the corresponding neck region with 70% ethanol to reduce the possibility of contamination.</li>
	<li>Using sterile scissors and forceps, aseptically dissect the proximal auricular dLN without collecting the surrounding fat. Perform a first incision in the jugulo-carotidian sulcus and remove the hairs from the operating field. Stretch the incision with 2 forceps to expose the dLN that appears grayish compared to the surrounding tissue.</li>
	<li>Pinch the connective tissues carefully on the top of the dLN with forceps to facilitate its removal. Extract the dLN by placing a forceps underneath the lymphoid tissue (Figure 3A and 3B). Place the dLN in a well containing 4.5 ml SM + 70 &#956;m cell strainer (Plate A).</li>
	<li>Harvest the entire inoculated ear using sterile sharp scissors and forceps (Figure 4A). Separate the dorsal and ventral aspects of the ear by pinching and spacing apart the cut ends with two forceps (Figure 4B-4E). Place them into a well containing 4.5 ml SM (Plate B) making sure both tissues are completely immersed in the medium. 
	NOTE: In order to improve the reliability of the experiment and increase the number of cells of interest, pool 2 injected ears or 2 dLNs from the same mouse per sample.</li>
	<li>Harvest and process in parallel the ears and dLNs of mice inoculated with either 1x DPBS or salivary gland extracts. Include 2 additional ear and lymph node samples harvested from a naive mouse for negative and single staining compensation controls.</li>
</ol>
5. Cell Isolation from Lymph Nodes
<ol>
	<li>Incubate the lymph nodes (Plate A) at 37 &#176;C + 5% CO2 for 15 min under gentle agitation. Add 10 mM final Ethylenediaminetetraacetic acid (EDTA) per well to neutralize collagenase and DNase activities. Perform the next steps on ice.</li>
	<li>Dissociate lymph nodes using the top end of a 5 ml syringe plunger. Press in circular motions against the strainer until all that remains is white connective tissues.</li>
	<li>Rinse the cell strainer twice with 500 &#956;l 1x DPBS + 2% Fetal bovine serum (FBS) + 2mM EDTA. Transfer entire volume of the well in a 15 ml tube.</li>
	<li>Rinse the well twice with 500 &#956;l 1x DPBS + 2% FBS + 2mM EDTA and transfer the volume into the 15 ml tube. Keep tubes on ice.</li>
</ol>
6. Cell Isolation from Skin of the Ear
<ol>
	<li>Incubate the ear leaflets (Plate B) at 37 &#176;C + 5% CO2 for 1 hr under gentle agitation. 
	NOTE: After 20 min of incubation, cut the ear leaflets with scissors into small pieces to facilitate tissue digestion and put the sample back in the incubator for the remaining 40 min. Single cells can be observed in the medium at the end of the incubation time.</li>
	<li>Add 10 mM final EDTA per well to neutralize collagenase and DNase activities. Perform the next steps on ice.</li>
	<li>Transfer the ear tissue fragments and the whole volume of medium in a new well containing a 70 &#956;m cell strainer using a 1ml pipette. Cut 2 to 3 mm from the end of the tip to collect the ear tissue fragments more easily.</li>
	<li>Dissociate ear tissue fragments using the top end of a 5 ml syringe plunger. Press in circular motions against the strainer until all that remains is white connective tissues.</li>
	<li>Rinse the cell strainers twice with 500 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA.</li>
	<li>Transfer the entire volume of the well into a 50 ml-tube through a 70 &#956;m cell strainer.</li>
	<li>Rinse the well with 500 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA and transfer the volume into the 50 ml tube. Keep the tube on ice. 
	NOTE: The different steps to isolate total cells from the skin and dLN tissues are summarized in Figure 5.</li>
</ol>
7. Count and Viability of Collected Cells
<ol>
	<li>Centrifuge the skin and dLN samples for 5 min at 450 x g at 4 &#176;C and discard the supernatant. Gently resuspend the pellet with 300 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA.</li>
	<li>Collect a small volume of each sample to count the total number of cells isolated from the skin and dLN using a hemocytometer. Add 0.04% trypan blue to determine the cell viability.</li>
</ol>
8. Blocking of Non Antigen-specific Binding
<ol>
	<li>Add 10 &#181;g/ml of unlabelled CD16/CD32 Fc-block antibody. Incubate 20 min at 4 &#176;C (can go longer).</li>
	<li>Add 2 ml of cold 1x DPBS + 2% FBS + 2 mM EDTA. Centrifuge the sample for 5 min at 450 x g at 4 &#176;C and discard the supernatant. Gently resuspend the pellet with 300 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA and maintain the samples on ice.</li>
</ol>
9. Staining Skin and Lymph Node Myeloid Cells
<ol>
	<li>Incubate previously blocked skin and dLN isolated cells with Live/Dead tracker (i.e. 4&#39;,6-diamidino-2-phenylindole or DAPI), anti-CD45 and anti-CD11b specific antibodies. 
	NOTES: In order to enrich the myeloid cell subpopulations of interest, especially rare ones, CD45+ cells and lymphocytes can be respectively purified from the skin or depleted from the dLN by magnetic bead facilitating cell sorting. Since most of the cells isolated from the dLN are CD45+, the use of anti-CD45 is not mandatory. Other markers can be included to identify myeloid cell subpopulations of interest by positive or negative-gating strategy. In this protocol, the myeloid cells recruited in the dLN were enriched by excluding CD8&#945;+ cells (the whole T cell compartment can be targeted by using anti-CD3 specific antibody).</li>
	<li>Incubate 30 min at 4 &#176;C in the dark. Add 500 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA, centrifuge the sample 5 min at 450 x g at 4 &#176;C and discard the supernatant. Repeat the wash step twice.</li>
	<li>Resuspend the samples with 300 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA. Transfer the entire volume in a FACS tube through a 35 &#181;m cell strainer. Rinse the filter with 100 &#956;l 1x DPBS + 2% FBS + 2 mM EDTA.</li>
	<li>Run the stained cells in a flow cytometer. Gate live single cells (DAPI-,FSC-W) to exclude dead cells and doublets. Plot CD45+CD11b+ events for the skin (Figure 6A) and (CD45+)CD8&#945;-CD11b+ events for the dLN (Figure 6B).</li>
</ol>

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The authors declare that they have no competing financial interests.

In the perspective of large-scale vaccination of humans using a whole sporozoite malaria vaccine, one of the main challenges to overcome is to develop optimized routes and methods of parasite administration to ensure successful immunization and protection24,25. In humans, the evaluation of protective efficacy mediated by live attenuated parasites (LAP) has been performed following natural mosquito bites2, as well as intradermal, subcutaneous25,26 and IV immunizations27. As reported in rodents28 and non-human primates25, IV administration of LAP induces stronger protective immune responses compared to the above-mentioned routes. Nevertheless, non-IV routes of administration using needle and syringe still remains more suitable for human mass vaccination and efforts are currently being made to optimize their protective efficacy.
Recent studies conducted in mice demonstrated, especially in the case of the ID route, that the deposition of a small volume of sporozoite suspension (range of 1 &#181;l) in multiple injection sites (4 points) significantly increased parasite infectivity compared to a single inoculation of parasites diluted in larger volumes (50 &#181;l)24. In our experimental system (C57BL/6 mouse - P. berghei), complete protection was achieved after multiple injections in the ear dermis of highly concentrated suspension of parasites (50,000 RAS in 0.6 &#181;l, 4 injection sites)19. In this context, we established the protocol described above to analyze more precisely the local host immune response to immunizing doses of RAS that allowed us to identify 2 major myeloid cell subsets recruited in the skin and dLN in response to the parasite19.
Multi-parametric flow cytometry allows accurate identification and quantification of immune cell changes in the context of host response to infection19,20. Isolation of large number of viable cells is therefore critical to perform reproducible phenotypic analysis. For this purpose, the enzyme concentration and the duration of tissue digestion must be optimized. Indeed, low concentration of enzymes or insufficient incubation time may lead to incomplete digestion with low yield of cells while prolonged tissue digestion may result in decrease cell viability and alteration of cell surface antigen expression29. Nevertheless, well-established protocols using collagenase treatment have been shown to have marginal impact on the detectability of relevant surface molecules frequently used for phenotypic analyses30.
Among the several parameters that can optimize the quality of cell isolation, the separation of the dorsal and ventral sections of the ear skin is essential, since it allows the collagenase/DNAse to access the dermis. In addition, mincing the skin into small pieces increases the surface area accessible to the enzymes, therefore allowing shorter duration of digestion. Finally, the use of mechanical dissociation helps to increase the yield in cell number for both the skin and dLN. However, excessive dissociation can cause additional stress that could negatively impact cell viability.
We favoured acquisition of live cells since fixation leads to suboptimal staining, complicating distinction of rare positive events of interest (data not shown). Moreover, the discrimination of dead cells was more problematic. In cases where cell fixation is required, we would suggest the use of LIVE/DEAD fixable dead cell stains.
Using the defined protocol, we successfully isolated myeloid cells infiltrating into the skin and dLN in response to parasite injection (Figure 6A and B). We usually obtain higher levels of viable single cells from the dLN (70-90%) compared to the skin (40-70%). This difference can be explained by the longer incubation time needed for efficient enzymatic digestion together with additional steps of mechanical dissociation to isolate skin cells (skin layer separation, mincing and stronger mashing).
Finally, an important parameter to take into account when analysing the host response to ID injection of sporozoites is the level of inflammation induced by both needle insertion and mosquito salivary gland extract deposition in the skin19,20. We therefore suggest to inject additional mice with either 1x PBS alone or salivary gland extracts from the same number of non-infected mosquitoes to evaluate the immune response specifically induced by the parasite. Altogether, we advise to dissect infected mosquitoes with high parasite load in the salivary glands and to filter the parasite suspension to limit the amount of mosquito material that could be deposited in the skin.
In summary, the isolation of myeloid cells from the skin and dLN through the described protocol provides a reliable assay to assess dynamic cellular changes during inflammatory response to Plasmodium infection and can be extended to other pathogens transmitted into the skin of the host.

Malaria is one of the deadliest infectious diseases in the world, killing more than half a million people per year. Infection by Plasmodium, the causal agent of the disease, begins by a pre-erythrocytic (PE) phase. During this phase, sporozoites injected into the host skin by a female Anopheline mosquito reach the liver via the bloodstream and differentiate inside hepatocytes into the parasite forms that infect red blood cells and cause the symptoms of the disease.
The PE stages of Plasmodium represent a privileged target for anti-malaria vaccination. Indeed, live attenuated vaccines against these stages, such as radiation attenuated sporozoites (RAS), genetically arrested parasites (GAP) or chemoprophylaxis and sporozoites (CPS) have demonstrated their capacity to protect both rodent and human hosts1-9. In the rodent model, most vaccination studies are conducted utilizing intravenous immunization, which is the gold standard in terms of protective efficacy. However, the description of a skin stage and the importance of the skin-associated draining lymph node (dLN) in eliciting protection has changed our perception of the PE phase and emphasized the importance of the intradermal route of injection. Intravital imaging of P. berghei sporozoites injected into the skin of rodents has shown that only ~25% of the inoculum reaches the liver via the bloodstream. The remaining ~75% distributes between the proximal dLN (~15%) and the skin (~50%)10,11, where a small proportion can transform and remain alive for weeks inside skin cells12,13. Moreover, subsequent studies described that the establishment of effective protective immunity after intradermal immunization mainly takes place in the skin-dLN, where parasite specific CD8+ T cells are activated, and only marginally in the spleen or liver-dLNs14,15.
While most studies have concentrated on the characterization of the effector cells implicated in the establishment of protective immune response, much less is known about the fate of live attenuated parasites injected into the skin, especially their interactions with the innate immune system. In particular, characterization of antigen-presenting cells involved in parasite antigen uptake, processing and presentation to CD8+ T cells is of critical importance, knowing that PE antigen acquisition can occur both in the skin and dLN compartments. Previous intravital imaging studies described an early influx of brightly fluorescent Lys-GFP positive cells in the skin following an infectious mosquito bite16 while early interactions between sporozoites and dendritic cells were observed in the dLN10,17. More recently, it has been reported that sporozoites inoculated in the skin by mosquitoes increases the motility of both dendritic and regulatory T cells in the skin of mice, while a decrease number of antigen presenting cells was observed in the dLN18.
We aimed to identify and quantify more precisely the leukocyte subsets recruited in the skin and corresponding dLN as well as those interacting with the parasite following intradermal injection of immunizing doses of RAS19. In this context, we isolated myeloid cells (CD45+CD11b+) from both tissues and characterized subpopulations of interest by multi=parametric flow cytometry. Consistent with the immune response described in the early stage of Leishmania major skin infection20, the primary host response to sporozoite injection consists of a successive recruitment of polymorphonuclear neutrophils (CD45+CD11b+Ly6G+Ly6Cint) followed by inflammatory monocytes (CD45+CD11b+Ly6G-Ly6C+) which are identified on the basis of differential expression of the Ly6G and Ly6C surface markers.
We describe here a protocol for isolating myeloid cells from the mouse skin and dLN following intradermal injection of immunizing doses of RAS extracted from infected mosquito salivary glands. Reproducible intradermal injections and tissue processing are critical steps to quantify phenotypic changes of infiltrating cell population within infected tissues. The approach detailed below provides a reliable assay to assess the skin and dLN inflammatory response to Plasmodium parasite and can be extended to various experimental systems.

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		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
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			<td>Xylazine: Rompun&#174; 2%</td>
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			<td></td>
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			<td></td>
			<td></td>
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			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
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			<td></td>
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			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
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			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
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			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
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			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
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			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
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			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
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			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
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			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
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			<td>Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C5138</td>
			<td>400 U/ml&#160;</td>
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			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
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			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
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			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
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			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Trypan blue Stain (0,4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Dilution 1:10&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32 (2.4G2 clone)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>10&#181;g/ml final (1:50)</td>
		</tr>
		<tr>
			<td>DAPI FluoroPureTM grade</td>
			<td>Life Technologies</td>
			<td>D21490</td>
			<td>1&#181;g/ml final</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (30-F11 clone)</td>
			<td>BD Biosciences</td>
			<td>559864</td>
			<td>&#160;Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (M1/70 clone)</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td>&#160;Dilution 1:400</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945; (5H10 clone)</td>
			<td>Life Technologies</td>
			<td>MCD0830</td>
			<td>&#160;Dilution 1:100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)&#160;</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

We recently demonstrated that needle-syringe injection of immunizing doses of P. berghei sporozoites in the mouse skin induces a successive recruitment of polymorphonuclear neutrophils followed by inflammatory monocytes in the skin and dLN19. The Protocol Section described above details the procedure used to successfully isolate live myeloid cells from both tissues following multiple injections of large number of sporozoites in the ear dermis (Figures 1 and 2). Within the first 24 hr, the dLN (Figure 3) and the skin injection site (Figure 4) were isolated and processed as summarized in Figure 5 to analyse the cellular infiltration by multi-parametric flow cytometry. Overall, this technique allowed us to isolate an average 104 CD45+CD11b+ and 2.104 CD11b+ live single cells for the skin and dLN respectively. The acceptable viability of non-debris single cells ranged from 40-70% for the skin and 70-90% for the dLN. As shown in Figure 6, the frequency of myeloid cells in the skin (CD45+CD11b+) and the dLN (CD8&#945;-CD11b+) strongly increases following intradermal injection of RAS compared with non-infected mice.
<img alt="Figure 1" src="/files/ftp_upload/53796/53796fig1.jpg" /> 
Figure 1: Intradermal Injection of Sporozoites into the Ear of Mouse. (A) Image showing intradermal injection of sporozoites in the ear pinna of an anesthetized mouse. The ventral side of the ear is stabilized with clear tape previously placed under a dissecting microscope. The needle is carefully inserted on the dorsal side of the ear, beneath the epidermis, with the bevel up. (B-C) Pictures showing the dorsal side of the ear pinna prior (B) and after (C) intradermal injection of 0.1-0.2 &#956;l of parasite suspension. A characteristic papule (black arrow) is observable at the injection site at the end of the operation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53796/53796fig2.jpg" /> 
Figure 2: Imaging of Sporozoites Deposited in the Ear Dermis of Mouse. (A) Fluorescence microscopy showing RAS migrating from the injection site (indicated by the dashed lines) in the mouse skin 15 min post-injection (Scale bar = 60 &#956;m; 10X magnification). The path of sporozoites is represented by the maximum intensity projection of the fluorescent signal. Green and red fluorescence respectively show parasite position in the skin at the beginning and after 10 sec of acquisition. (B) Higher magnification of RAS (green) injected in the skin (Scale bar = 20 &#956;m; 25X magnification). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53796/53796fig3.jpg" /> 
Figure 3: Draining Lymph Node Processing. (A) Picture showing the dissection of the proximal auricular dLN following intradermal injection of Evans blue for demonstration purposes. After cervical dislocation of the mouse, the neck is disinfected with 70% ethanol. A first incision is made in the jugular-carotid area with sharp scissors and the skin is removed from the operating field. The incision is stretched with 2 forceps to expose the dLN that appears grayish compared to the surrounding tissue. The connective tissues are then carefully pinched with forceps on the top of the dLN and progressively separated from it. Finally, the dLN is extracted by placing a forceps underneath the lymphoid tissue. (B) Picture showing the extracted dLN in a drop of PBS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53796/53796fig4.jpg" /> 
Figure 4: Ear Skin Processing. Pictures showing the successive steps to process the inoculated ear. (A) After cervical dislocation of the mouse, the ear is disinfected with 70% ethanol and removed using sterile sharp scissors and forceps. (B-D) The dorsal and ventral aspects of the ear are lightly separated by pinching and spacing apart the cut ends with two forceps. (E) Picture showing the dorsal and ventral aspects of the ear prior enzymatic treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53796/53796fig5.jpg" /> 
Figure 5: Protocol Summary. Schematic representation of the key steps to isolate total cells from the skin and dLN tissues. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53796/53796fig6.jpg" /> 
Figure 6: Myeloid Cell Population Isolated From the Ear Skin and the Proximal dLN. Representative FACS plots showing the gating strategy to analyse the myeloid compartment in the skin (A) and the dLN (B). CD45+CD11b+ and CD8&#945;-CD11b+ cells were gated on total live singlet events for the skin and dLN respectively. For each condition, 5 x 105 total events were acquired (2 ears and 2 dLN pooled per sample). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53796/53796fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

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.

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 ...

myeloid-cell-isolation-from-mouse-skin-draining-lymph-node-following

Nanyang Technological University

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JoVE Journal

Immunology and Infection

17.1K Views.  Institut Pasteur. 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.

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

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

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, ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...