Name: a simple protocol for extracting hemocytes from wild caterpillars
Uploaded: 2012-11-15T11:00:00
Description: Watch this Scientific Journal Video about A Simple Protocol for Extracting Hemocytes from Wild Caterpillars at JoVE.com

TMS ble støttet av Harlan Trust Fellowship (GWU) i denne studien. Midler til feltet innsamling og avl L. fasciola og E. delphinii ble levert av NSF stipend DEB 0642438 til JTL.

1. Materiale Forberedelse <ol><li> Forbered nåler (1-2 mikrometer tips og 3-4 mm taper) ved hjelp av borosilicate glass kapillærrør og en mikropipette avtrekker. Instrumentinnstillinger: rampe: 561; hastighet: 20; varme: 560; forsinkelse: 1; pull: 100; trykk: 500. </li><li> Klargjør samlingen løsning: 60% av Grace Medium (GM) supplert med 10% av føtalt bovint serum (FBS) og 20% ​​av Antikoagulerende Buffer (98 mM NaOH, 186 mM NaCl, 1,7 mM EDTA og 41 mM sitronsyre, pH 4,5 ). Den ovennevnte løsning er forbere...

<ol>
	<li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+blood+cell+development+in+the+animal+kingdom.">Blood cells and blood cell development in the animal kingdom.</a> Annu. Rev. Cell Dev. Biol. 22, 677 (2006).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+embryonic+haemocytes:+masters+of+multitasking.">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat. Rev. Mol. Cell Biol. 8 (7), 542 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15 (1), 1 (2008).</li><li>Fauvarque, M. O., Williams, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+cellular+immunity:+a+story+of+migration+and+adhesion.">Drosophila cellular immunity: a story of migration and adhesion.</a> J. Cell Sci. 124 (9), 1373 (2011).</li><li>Nehme, N. T., Quintin, J., Cho, J. H., Lee, J., Lafarge, M. C., Kocks, C., Ferrandon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+the+cellular+and+humoral+responses+in+the+Drosophila+host+defense+against+three+gram-positive+bacterial+infections.">Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections.</a> PLoS One. 6 (3), e14743 (2011).</li><li>Ulvila, J., Vanha-Aho, L. M., R&#228;met, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+phagocytosis+-+still+many+unknowns+under+the+surface.">Drosophila phagocytosis - still many unknowns under the surface.</a> APMIS. 119 (10), 651 (2011).</li><li>Moreira, C. G., Regan, J. C., Zaidman-Remy, A., Jacinto, A., Praq, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hemocyte+migration:+an+in+vivo+assay+for+directional+cell+migration.">Drosophila hemocyte migration: an in vivo assay for directional cell migration.</a> Methods Mol. Biol. 769, 249 (2011).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+immunity.">Mosquito immunity.</a> Adv. Exp. Med. Biol. 708, 218 (2010).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36 (12), 891 (2006).</li><li>Jiang, H., Vilcinskas, A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+in+lepidopteran+insects.">Immunity in lepidopteran insects.</a> Adv. Exp. Med. Biol. 708, 181 (2010).</li><li>Eleftherianos, I., Xu, M., Yadi, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmatocyte-spreading+peptide+(PSP)+plays+a+central+role+in+insect+cellular+immune+defenses+against+bacterial+infection.">Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.</a> J. Exp. Biol. 212 (12), 1840 (2009).</li><li>Ribeiro, C., Breh&#233;lin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+haemocytes:+what+type+of+cell+is+that.">Insect haemocytes: what type of cell is that.</a> J. Insect Physiol. 52 (5), 417 (2006).</li><li>Beetz, S., Brinkmann, M., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+larval+and+pupal+hemocytes+of+the+tobacco+hornworm,+Manduca+sexta,+determined+by+monoclonal+antibodies+and+density+centrifugation.">Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation.</a> J. Insect Physiol. 50 (9), 805 (2004).</li><li>Beetz, S., Holthusen, T. K., Koolman, J., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+hemocyte+counts+with+different+developmental+parameters+during+the+last+larval+instar+of+the+tobacco+hornworm,+Manduca+sexta.">Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 67 (2), 63 (2008).</li><li>Eleftherianos, I., Joyce, S., ffrench-Constant, R. H., Clarke, D. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+tri-trophic+interaction+between+insects,+nematodes+and+Photorhabdus.">Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.</a> Parasitology. 137 (11), 1695 (2010).</li><li>Qayum, A. A., Telang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Protocol+for+Collecting+and+Staining+Hemocytes+from+the+Yellow+Fever+Mosquito+Aedes+aegypti.">A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti.</a> J. Vis. Exp. (51), e2772 (2011).</li></ol>

Metoder for å trekke hemocytes fra medisinsk viktige insekter og lepidopteran modell insekter har tidligere blitt rapportert 9, 14. Hemocyte ekstraksjonsmetoder metoder tilpasses studentens insektarter, utviklingsstadiet av insekt og dens morfologiske egenskaper. For eksempel, kan hemolymph isolasjon fra Manduca larver lett utføres ved snipping den buede hornet nær slutten av buken 15. Fordi slug sommerfugllarver (Limacodidae) mangler denne abdominal strukturen, har vi utviklet en disti...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Mikropipette avtrekker </td><td> Sutter Instruments </td><td> P-1000 </td><td></td></tr><tr><td> Borsilikatglass rør </td><td> Sutter Instruments </td><td> B100-50-10 </td><td> OD: 1.0, ID: 0,50 mm </td></tr><tr><td> Grace Medium </td><td> Sigma </td><td> G8142 </td><td></td></tr><tr><td> Føtalt bovint serum </td><td> Thermo Fisher Scientific </td><td> SH3007102 </td><td> Varmeinaktivert </td></tr><tr><td> Dumont # 5 tang </td><td> Fin vitenskap verktøy </td><td> 11252-40 </td><td></td></tr><tr><td> Neubauer hemocytometer </td><td> Häusser Scientific </td><td> 3200 </td><td></td></tr><tr><td> Plast TuBing </td><td> Tri-Tech </td><td> TT-3-32OD </td><td> OD: 3/32&#39;&#39;, ID: 1/32 &#39; </td></tr><tr><td> Glass medisinske sprøyte </td><td> Fortuna Optima </td><td> D-97877 </td><td> 50 ml volum </td></tr><tr><td> Butte enden nål </td><td> Små deler </td><td> NE-162PL-25 </td><td> 16 Gauge x 1 &quot;lengde </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

Insect hemocytes (tilsvarer mammalske hvite blodlegemer) spiller en viktig rolle i flere fysiologiske prosesser gjennom et insekt livssyklus 1. I larvestadier av insekter som tilhører ordre fra Lepidoptera (møll og sommerfugler) og tovinger (sanne fluer), er hemocytes dannet fra lymph kjertel (en spesialisert blodkreft organ) eller embryonale celler og kan bli gjennomført til det voksne stadiet. Embryonale hemocytes er involvert i celle migrasjon under utvikling og chemotaxis regulering ved betennelse. De tar også del i celle apoptose og er avgjørende for embryogenese 2. Hemocytes megle cellenivå arm av insekt medfødte immunforsvaret som inkluderer flere funksjoner, for eksempel celle spredning, celle aggregering, dannelsen av knuter, fagocytose og innkapsling av utenlandske inntrengere 3. De er også ansvarlig for iscenesetter spesifikke insekt humorale forsvar under infeksjonen, som produksjon av antimikrobielle peptider og andre effektor molekyler 4, 5. Hemocyte morfologi og funksjon er i hovedsak blitt studert i genetiske eller fysiologisk insekt modeller, inkludert bananflue, Drosophila 6 melanogaster, 7, myggen Aedes aegypti og Anopheles gambiae 8, 9 og tobakk hornworm, Manduca 10 sexta, 11. Det finnes imidlertid lite informasjon foreløpig om mangfold, klassifisering, morfologi og funksjon av hemocytes i ikke-modell insektarter, særlig de hentet fra naturen 12. Her beskriver vi en enkel og effektiv protokoll for å trekke hemocytes fra ville larvene. Vi bruker nest siste stadium Lithacodes fasciola (gul-shouldered slug møll) (figur 1) og Euclea delphinii (spiny eik slug) sommerfugllarver (Lepidoptera: Limacodidae) og viser at tilstrekkelige mengder hemolymph (insekt blod)kan bli isolert og hemocyte numre regnet fra individuell larver. Denne metoden kan brukes til å effektivt studere hemocyte typer i disse artene samt i andre relaterte lepidopteran sommerfugllarver høstes fra feltet, eller den kan lett kombineres med immunologiske analyser for å undersøke hemocyte funksjon etter infeksjon med mikrobiell eller parasittiske organismer 13.

Protokollen beskrevet heri tillater innsamling av et minimum volum på 10-20 pl insektvoks hemolymph fra individuelle larvene. Hemocytes samlet ved hjelp av denne metoden er fri for celle klumper, melanization defekter, vev rusk eller andre forurensninger. Derfor hemocytes kan lett observeres og telles under mikroskop, og flere insekter kan observeres i løpet av få Hosur. Vi observerte at flertallet av hemocytes i våre prøver besto av plasmatocytes 3 ​​(hemocytes sprer asymmetrisk) (figur 4).<...

Watch this Scientific Journal Video about A Simple Protocol for Extracting Hemocytes from Wild Caterpillars at JoVE.com

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insekt hemocytes utføre mange viktige funksjoner, både immune og ikke-immune, gjennom alle stadier av insekter utvikling. Vår nåværende kunnskap om hemocyte typer og funksjon kommer fra studier på insekt genetiske modeller. Her presenterer vi en metode for å trekke ut, kvantifisere og visualisere hemocytes fra ville larvene.

En enkel protokoll for å trekke Hemocytes fra Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Research

JoVE Journal

Biology

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

Protocol for Mosquito Rearing (A. gambiae)

Protocol for Mosquito Rearing A. gambiae

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

En PCR-basert genotyping metode for å skille mellom vill-type og Ornamental Varianter av Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM-protokollen for ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

En enkel Bioassay for Evaluering av vaskulær endotelial vekstfaktorer

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Filtrering Isolering av nukleinsyrer: En enkel og Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Method for Extracting Pigments from Squid Doryteuthis pealeii

En metode for utvinning av Pigmenter fra Squid<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...