Name: isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice
Uploaded: 2013-01-02T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Forfatterne vil gerne takke Miss Liana Mackiewicz til teknisk bistand. Dette arbejde blev gjort mulig gennem Victorian delstatsniveau operationelle infrastruktur Support og australske regering National Health og Medical Research Council IRIISS og Project Grant 1.031.212.

1. Infektion af mus med P. berghei-ANKA <ol><li> Afrimning en alikvot af kryopræserveret P. berghei ANKA PRBC. </li><li> Fastholde en cerebral malaria-resistent BALB / c donormus (8-12 uger gamle) ved hjælp af begge hænder fastholdelsesanordning teknik. Sprøjt mus med 100-200 ml PRBC anvendelse af en 1 ml insulinsprøjte (28G nål). Rutinemæssigt 1-2 donormus injiceret. </li><li> På dage 4-5 post-infektion (pi) fjerne donor mus fra bur og placere den på en arbejdsstation eller en engangs arbej...

<ol>
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Isoleringen og analyse af BSL er en fremgangsmåde, som muliggør karakterisering og kvantificering af inflammatoriske celler migrerer til hjernen som reaktion på vævsbeskadigelse eller infektion i eksperimentelle musemodeller. Indførelsen af ​​en intracardial perfusion trin til fjernelse af blodfordelingsrummet før organ-ekstraktion og efterfølgende celleisolering er nyttig for at forhindre forurening af inflammatoriske celler med ikke-inflammatoriske cirkulerende leukocytter. Dette er måske ikke et væsentli...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td></td><td></td><td></td><td> Løsninger og buffere </td></tr><tr><td> Giemsa s azur eosin opløsning af methylenblåt </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> 1:10 fortynding i destilleret vand </td></tr><tr><td> RPMI-medium </td><td></td><td></td><td> Mus toniciteten </td></tr><tr><td> Mus toniciteten PBS </td><td></td><td></td><td> 20 mM natriumphosphat, 0,149 NaCl, pH 7,3 </td></tr><tr><td> 0,4% Trypan Blue </td><td> Sigma Aldrich </td><td> T-8154 </td><td> 1:2 fortynding </td></tr><tr><td> Collagenase D </td><td> Worthington Biochemical </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAse) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> Fra bovin bugspytkirtel </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 30% opløsning i PBS </td></tr><tr><td> Ultrarent Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Ammoniumchlorid (NH4CI) </td><td> AnalaR </td><td> 10.017 </td><td></td></tr><tr><td> Red Cell Lysis Buffer </td><td></td><td></td><td> 17 mM Tris, 14nM NH4CI, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA-dinatriumsalt </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Antistoffer og konjugater </td></tr><tr><td> Anti-muse CD16/CD32 (Fc Block), klon 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553.142 </td><td> 1 pi i 50 pi farvning puffer (0,5 mg/50 ml) </td></tr><tr><td> FITC-anti-muse CD4, klon H129.19 </td><td> BD Pharmingen </td><td> 553.651 </td><td></td></tr><tr><td> PE-anti-muse NK1.1, klon PK136 </td><td> BD Pharmingen </td><td> 553.165 </td><td></td></tr><tr><td> PerCPCy5.5-anti-muse-CD8, klon 53-6-7 </td><td> BD Pharmingen </td><td> 551.162 </td><td></td></tr><tr><td> APC-anti-muse-TCR-β, klon H57-597 </td><td> BD Pharmingen </td><td> 553.174 </td><td></td></tr><tr><td> PE-anti-muse CXCR3, klon 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinyleret-anti-muse-CCR5, klon C34-3448 </td><td> BD Pharmingen </td><td> 559.922 </td><td></td></tr><tr><td> Streptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551.419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Udstyr og materiel </td></tr><tr><td> SuperFrost mikroskopobjektglas </td><td> Lomb Menzel-Gläser </td><td></td><td></td></tr><tr><td> Dissektionsværktøj pincet, saks </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml PBS reservoir </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Gummislanger </td><td></td><td></td><td></td></tr><tr><td> 23G nål </td><td> BD PrecisionGlide </td><td> 302.008 </td><td></td></tr><tr><td> Celledissociering kit indeholdende metalsi </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 pm nylon cell strainer </td><td> BD Falcon </td><td> 352.350 </td><td></td></tr><tr><td> Hæmocytometer </td><td> GmbH Neubauer </td><td> 717.810 </td><td></td></tr><tr><td> Flowcytometri rør </td><td>BD Falcon </td><td> 352.008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Vi beskriver en fremgangsmåde til isolering og karakterisering af adhærente inflammatoriske celler fra hjernen blodkar P. berghei ANKA-inficerede mus. Infektion af modtagelige muse-stammer med denne parasit stamme resulterer i induktion af eksperimentel cerebral malaria, en neurologisk syndrom, der redegør for en række vigtige aspekter af Plasmodium falciparum-medieret alvorlig malaria hos mennesker 1,2. Modne former af blod-stage malaria udtrykker parasitiske proteiner på overfladen af ​​den inficerede erythrocyt, som tillader dem at binde til vaskulære endotelceller. Denne proces inducerer forhindringer i blodstrømmen, hvilket resulterer i hypoxi og blødninger 3 og også stimulerer rekruttering af inflammatoriske leukocytter til stedet for parasitten binding. I modsætning til andre infektioner, dvs neutrotopic vira 4-6, både malaria-parasitized røde blodlegemer (PRBC) samt tilhørende InflammBAGGRUNDEN leukocytter forbliver sekvestreres i blodkarrene i stedet for at infiltrere hjerneparenkymet. Således at undgå forurening af sekvestrerede leukocytter med ikke-inflammatoriske cirkulerende celler, omfattende intracardial perfusion af inficerede-mus forud for organdonation og væv behandling er påkrævet i denne procedure for at fjerne blod rum. Efter perfusion bliver hjerner høstet og dissekeret i små stykker. Vævsstrukturen yderligere afbrudt ved enzymatisk behandling med collagenase D og DNAse I. Det resulterende hjernehomogenat centrifugeres derefter på en Percoll-gradient, som tillader separation af hjerne-sekvestrerede leukocytter (BSL) fra myelin og andet væv debris. Isolerede celler vaskes derefter, talt under anvendelse af et hæmocytometer og farvet med fluorescerende antistoffer til efterfølgende analyse ved flowcytometri. Denne procedure muliggør omfattende fænotypisk karakterisering af inflammatoriske leukocytter migrerer til hjernen i respons på forskellige stimuli, herunder slagtilfælde såvel som virale eller parasitiske infektioner. Fremgangsmåden tilvejebringer også et nyttigt værktøj til vurdering af nye antiinflammatoriske behandlinger i prækliniske dyremodeller.

Resultaterne i fig. 2 viser procenter og absolutte tal af forskellige BSL populationer udvundet fra hjerner af perfunderede eller unperfused malaria-inficerede og naive kontrolmus. Isoleret BSL blev farvet med PE-anti-NK1.1 og APC-anti-TCR-β-antistoffer er angivet i protokollen teksten. I overensstemmelse med tidligere resultater 7-9, αβTCR + T-celler omfatter en stor del af BSL pool i hjernen hos perfunderede malaria-inficerede mus (dag 6 pi). Denne population syntes at være betydeligt underre...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Fremgangsmåde til isolering af adhærente inflammatoriske leukocytter fra hjernen blodkar<em&gt; Plasmodium berghei</em&gt; ANKA-inficerede mus er beskrevet. Fremgangsmåden tillader kvantificering og fænotypiske karakterisering af isolerede leukocytter efter farvning med fluorescerende antistoffer og efterfølgende analyse ved flowcytometri.

Isolering og analyse af Brain-sekvestrerede Leukocytter fra<em&gt; Plasmodium berghei</em&gt; ANKA-inficerede mus

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

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An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

En eksperimentel model til at studere tuberkulose-Malaria coinfektion på Natural Transmission af Mycobacterium tuberculosis Og Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

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Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

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At analysere virkningen af ​​stromacellers om ansættelse af Leukocytter fra Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

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Isolering af leukocytter fra Human Maternal-føtal grænseflade

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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Isolering af leukocytter fra det murine væv på maternel-føtal grænseflade

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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

Isolering af leukocytter fra musehud anvendelse af enzymatiske Digest og Gradient Separation

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolering og flowcytometrisk analyse af immunceller fra iskæmisk Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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

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

Myeloid Cell Isolation fra Mouse Hud og drænende lymfeknude Efter Intrakutan Vaccination med levende svækket<em&gt; Plasmodium</em&gt; sporozoitter

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

Selective Harvesting of Marginating-pulmonary Leukocytes

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Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...