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 <e...

<ol>
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K., 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=Plasmodium+liver+stage+developmental+arrest+by+depletion+of+a+protein+at+the+parasite-host+interface.">Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface.</a> Proc Natl Acad Sci U S A. 102 (8), 3022-3027 (2005).</li><li>Mueller, A. K., Deckert, M., Heiss, K., Goetz, K., Matuschewski, K., Schl&#252;ter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+attenuated+Plasmodium+berghei+liver+stages+persist+and+elicit+sterile+protection+primarily+via+CD8+T+cells.">Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells.</a> Am J Pathol. 171 (1), 107-115 (2007).</li><li>Tarun, A. 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=Protracted+sterile+protection+with+Plasmodium+yoelii+pre-erythrocytic+genetically+attenuated+parasite+malaria+vaccines+is+independent+of+significant+liver-stage+persistence+and+is+mediated+by+CD8++T+cells.">Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells.</a> J Infect Dis. 196 (4), 608-616 (2007).</li><li>van Dijk, M. R., 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=Genetically+attenuated,+P36p-deficient+malarial+sporozoites+induce+protective+immunity+and+apoptosis+of+infected+liver+cells.">Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.</a> Proc Natl Acad Sci U S A. 102 (34), 12194-12199 (2005).</li><li>Butler, N. S., Schmidt, N. W., Vaughan, A. M., Aly, A. S., Kappe, S. H., Harty, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+antimalarial+immunity+after+vaccination+with+late+liver+stage-arresting+genetically+attenuated+parasites.">Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites.</a> Cell Host Microbe. 9 (6), 451-462 (2011).</li><li>Belnoue, E., 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=Protective+T+cell+immunity+against+malaria+liver+stage+after+vaccination+with+live+sporozoites+under+chloroquine+treatment.">Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment.</a> J Immunol. 172 (4), 2487-2495 (2004).</li><li>Behet, M. C., 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=Sporozoite+immunization+of+human+volunteers+under+chemoprophylaxis+induces+functional+antibodies+against+pre-erythrocytic+stages+of+Plasmodium+falciparum.">Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum.</a> Malar J. 13, 136 (2014).</li><li>Amino, R., 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=Quantitative+imaging+of+Plasmodium+transmission+from+mosquito+to+mammal.">Quantitative imaging of Plasmodium transmission from mosquito to mammal.</a> Nat Med. 12 (2), 220-224 (2006).</li><li>Yamauchi, L. M., Coppi, A., Snounou, G., Sinnis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+sporozoites+trickle+out+of+the+injection+site.">Plasmodium sporozoites trickle out of the injection site.</a> Cell Microbiol. 9 (5), 1215-1222 (2007).</li><li>Gueirard, 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=Development+of+the+malaria+parasite+in+the+skin+of+the+mammalian+host.">Development of the malaria parasite in the skin of the mammalian host.</a> Proc Natl Acad Sci U S A. 107 (43), 18640-18645 (2010).</li><li>Voza, T., Miller, J. L., Kappe, S. H., Sinnis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrahepatic+exo-+erythrocytic+forms+of+rodent+malaria+parasites+at+the+site+of+inoculation:+clearance+after+immunization,+susceptibility+to+primaquine,+and+contribution+to+blood-stage+infection.">Extrahepatic exo- erythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection.</a> Infect Immun. 80 (6), 2158-2164 (2012).</li><li>Chakravarty, S., Cockburn, I. A., Kuk, S., Overstreet, M. G., Sacci, J. B., Zavala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+lymphocytes+protective+against+malaria+liver+stages+are+primed+in+skin-draining+lymph+nodes.">CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes.</a> Nat Med. 13 (9), (2007).</li><li>Obeid, M., 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=Skin-draining+lymph+node+priming+is+sufficient+to+induce+sterile+immunity+against+pre-erythrocytic+malaria.">Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.</a> EMBO Mol Med. 5 (2), 250-263 (2013).</li><li>Amino, R., 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=Host+cell+traversal+is+important+for+progression+of+the+malaria+parasite+through+the+dermis+to+the+liver.">Host cell traversal is important for progression of the malaria parasite through the dermis to the liver.</a> Cell Host Microbe. 3 (2), 88-96 (2008).</li><li>Radtke, A. J., 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=Lymph-node+resident+CD8&#945;++dendritic+cells+capture+antigens+from+migratory+malaria+sporozoites+and+induce+CD8++T+cell+responses.">Lymph-node resident CD8&#945;+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.</a> PLoS Pathog. 11 (2), e1004637 (2015).</li><li>da Silva, H. B., 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=Early+skin+immunological+disturbance+after+Plasmodium-infected+mosquito+bites.">Early skin immunological disturbance after Plasmodium-infected mosquito bites.</a> Cell Immunol. 277 (1-2), 22-32 (2012).</li><li>Mac-Daniel, L., 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=Local+immune+response+to+injection+of+Plasmodium+sporozoites+into+the+skin.">Local immune response to injection of Plasmodium sporozoites into the skin.</a> J Immunol. 193 (3), 1246-1257 (2014).</li><li>Ribeiro-Gomes, F. L., Peters, N. C., Debrabant, A., Sacks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+capture+of+infected+neutrophils+by+dendritic+cells+in+the+skin+inhibits+the+early+anti-leishmania+response.">Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response.</a> PLoS Pathog. 8 (2), e1002536 (2012).</li><li>Thiberge, 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=In+vivo.+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo. imaging of malaria parasites in the murine liver.</a> Nat Protoc. 2 (7), 1811-1818 (2007).</li><li>Ishino, T., Orito, Y., Chinzei, Y., Yuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-dependent+protein+kinase+regulates+Plasmodium+ookinete+access+to+the+midgut+epithelial+cell.">A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell.</a> Mol Microbiol. 59 (4), 1175-1184 (2006).</li><li>Amino, R., 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=Imaging+malaria+sporozoites+in+the+dermis+of+the+mammalian+host.">Imaging malaria sporozoites in the dermis of the mammalian host.</a> Nat Protoc. 2 (7), 1705-1712 (2007).</li><li>Ploemen, I. 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E., 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=Live+attenuated+malaria+vaccine+designed+to+protect+through+hepatic+CD8+T+cell+immunity.">Live attenuated malaria vaccine designed to protect through hepatic CD8 T cell immunity.</a> Science. 334 (6055), 475-480 (2011).</li><li>Roestenberg, M., 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=Long-term+protection+against+malaria+after+experimental+sporozoite+inoculation:+an+open-label+follow-up+study.">Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.</a> Lancet. 377 (9779), 1770-1776 (2011).</li><li>Seder, R. 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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 repo...

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.

<table>
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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 </stron...

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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 ...

The overall goal of this set of procedures is to immunize mice with radiation attenuated plasmodium parasites. To facilitate the analysis of myeloid cell recruitment in the mouse skin and draining lymph nodes. This method can help answer key questions in the malaria field, such as what are the cellular actors involved in the innate immunoresponse to live attenuated parasites.Between 18-25 days after the infections blood meal, collect and cold anesthetize infected mosquitoes in a 15 milliliter tube on ice. Next, carefully invert the tube into a Petri dish on ice and expose the insects to a 12 kilorad dose of gamma or X-irradiation. Collect the irradiated mosquito salivary glands in 15 microliters of DPBS on ice.And then, gently crush the glands to release the sporozoites. The injection of a highly concentrated parasite suspension in a small volume is critical, so it is important to harvest a sufficient number of salivary glands from well-infected mosquitoes as evidenced by robust expression of green fluorescence. Re-suspend the parasite suspension prior to filtration through a 35 micrometer strainer adapted to a 1.5 milliliter microcentrifuge tube.Using a microscope counting chamber, count the total number of isolated sporozoites and adjust the parasite suspension to an 8.3 times ten to the fourth sporozoites per microliter concentration in cold DPBS on ice. Before injecting the sporozoites, confirm the proper level of sedation of the experimental animal by toe pinch and apply eye ointment to prevent corneal drying. Then, working quickly, use clear tape to gently fix the ventral side of the ear pinna under a stereo microscope and load a ten microliter syringe, equipped with a 35 gauge needle, with 0.6 microliters of resuspended parasites.Next, carefully insert the needle, bevel side up, beneath the epidermis on the dorsal side of the ear and inject 0.15 microliters of sporozoites into the site. A characteristic papule will be observed at the injection site. After injecting the dermis three more times, as just demonstrated, carefully remove the tape and monitor the mouse until it is fully recovered.At the desired time point following sporozoite inoculation, sacrifice the mouse and start the procedure immediately. Make sure the mouse demonstrates signs of clinical death. Disinfect the ear and the corresponding neck region with 70 percent ethanol.Next, using sterile scissors and forceps, make an incision in the jugulo-carotidian area. Then stretch the incision with two forceps to expose the grey-looking draining lymph node. Carefully pinch and remove the connective tissue directly above the lymph node and extract it with a second pair of forceps, placed underneath it.Transfer the lymph node into a 70 micron strainer in one well of a six-well tissue culture plate, containing 4.5 milliliters of standard medium, supplemented with collagenase and DNase. Next, harvest the entire inoculated ear and use two forceps to pinch and separate the ear into the dorsal and ventral aspects. Then place each side of the ear into a well of a second six-well plate, containing 4.5 milliliters per well of medium supplemented with digestive enzymes, taking care that each tissue is completely immersed in the medium.To isolate the immune cells from the lymph nodes, incubate the plate of lymphoid tissue at 37 degrees Celsius and five percent CO2 under gentle agitation. After 15 minutes, add ten millimolar EDTA to each well and place the plate on ice. Next, for each lymph node, use the end of a new five millimeter syringe plunger and press the lymph node against the strainer using circular motions to dissociate the tissue.When only white connective tissue is left, rinse the strainer two times with 500 microliters of DPBS containing FBS and EDTA and transfer the wash to a 15 milliliter conical tube. Then, wash the well two more times with 500 microliters of the supplemented DPBS and transfer the wash to the tube placed on ice. To isolate the immune cells from the ear, incubate the ear leaflets at 37 degrees Celsius and five percent CO2 with gentle agitation.After 20 minutes, cut the tissues into smaller pieces to facilitate their digestion and return the samples to the incubator. After 40 minutes, stop the digestion with ten millimolar EDTA and place the plate on ice. Using a one milliliter pipette with a cut tip, transfer the entire contents of each well into new wells containing 70 micron strainers.And use a five milliliter syringe plunger to dissociate the ear tissue fragments with circular motions as just demonstrated. When only white connective tissue and hair remain, wash the strainers and wells with DPBS plus FBS and EDTA as just demonstrated, pooling the washes into a 50 milliliter tube on ice. Finally, wash the well twice with supplemented DPBS and transfer the wash to the tube placed on ice.In this video, the migration of radiation attenuated sporozoites into the mouse skin 15 minutes after intradermal injection of the parasites can be seen. 24 hours after intradermal sporozoite injection, total cells are isolated from the skin and draining lymph node. As observed on this figure, the frequency of myeloid cells in the skin and draining lymph node is robustly increased compared to naive mice.Once mastered, the first steps of the protocol for mosquito irradiation to sporozoite injection can be completed in four hours. While attempting this procedure, it is important to remember that sporozoite isolated from mosquito salivary glands rapidly lose their infectivity. So they must be injected as quickly as possible.After watching this video, you should have a good understanding of how to isolate myeloid cells from the mouse skin and draining lymph node to explore the immunoresponse to intradermal injection of live attenuated plasmodium parasites.

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Immunology and Infection

16.8K visualizações.  Institut Pasteur. The overall goal of this set of procedures is to immunize mice with radiation attenuated plasmodium parasites. To facilitate the analysis of myeloid cell recruitment in the mouse skin and draining lymph nodes. This method can help answer key questions in the malaria field, such as what are the cellular actors involved in the innate immunoresponse to live attenuated parasites.Between 18-25 days after the infections blood meal, collect and cold anesthetize infected mosquitoes in a 15 mil...