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 blev støttet af Harlan Trust Fellowship (GWU) under denne undersøgelse. Finansieringen af feltet indsamling og avl L. Fasciola og E. delphinii blev leveret af NSF tilskud DEB 0642438 til JTL.

1. Klargøring <ol><li> Forbered nåle (1-2 um spids og 3-4 mm taper) ved hjælp af borosilikatglas kapillarrør og en mikropipette aftrækker. Instrument indstillinger: Rampe: 561, Hastighed: 20; varme: 560, delay: 1; træk: 100; tryk: 500. </li><li> Forberede samlingen opløsning: 60% af Graces medium (GM) suppleret med 10% af føtalt bovint serum (FBS) og 20% ​​af antikoagulerende puffer (98 mM NaOH, 186 mM NaCl, 1,7 mM EDTA og 41 mM citronsyre, pH 4,5 ). Ovennævnte opløsning fremstilles frisk under sterile b...

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Fremgangsmåder til ekstraktion hæmocytter fra medicinsk vigtige insekter og lepidoptera-insekter model er tidligere blevet rapporteret, 9, 14. Hemocyte ekstraktionsmetoder er tilpasset efter de insektarter, udviklingsstadium insektet og morfologiske egenskaber. For eksempel kan hæmolymfe isolation fra Manduca larver let udføres ved at klippe det krumme horn nær enden af abdomen 15. Fordi slug larver (Limacodidae) mangler denne abdominal struktur, har vi udviklet en særskilt protokol ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Mikropipette aftrækker </td><td> Sutter Instruments </td><td> P-1000 </td><td></td></tr><tr><td> Borsilikat glasrør </td><td> Sutter Instruments </td><td> B100-50 til 10 </td><td> OD: 1,0, ID: 0,50 mm </td></tr><tr><td> Graces 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> Varmeinaktiveret </td></tr><tr><td> Dumont # 5 Tang </td><td> Fin science-værktøjer </td><td> 11.252-40 </td><td></td></tr><tr><td> Neubauer hæmocytometer </td><td> Hausser Scientific </td><td> 3200 </td><td></td></tr><tr><td> Plastic 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> Glas medicinsk sprøjte </td><td> Fortuna Optima </td><td> D-97877 </td><td> 50 ml volumen </td></tr><tr><td> Stumpe ende nål </td><td> Små Parts </td><td> NE-162PL-25 </td><td> 16 Gauge x 1 &quot;længde </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

Insekt hæmocytter (ækvivalent med mammale hvide blodlegemer) spiller en vigtig rolle i flere fysiologiske processer gennem en insektets livscyklus 1. I larvestadier af insekter hører til ordenerne Lepidoptera (møl og sommerfugle) og Diptera (ægte fluer), er hæmocytter dannet af lymfekirtel (en specialiseret hæmatopoietisk organ) eller embryonale celler og kan føres igennem til det voksne stadie. Embryoniske hæmocytter er involveret i cellemigrering under udvikling og kemotaxi regulering under inflammation. De tager også del i celle apoptose og er afgørende for embryogenese 2. Hæmocytter medierer den cellulære arm af insekt medfødte immunreaktion, der omfatter flere funktioner, såsom cellespredning, celleaggregering, dannelse af knuder, fagocytose og indkapsling af fremmede angribere 3. De er også ansvarlige for orkestrere særlige insekt humorale forsvar under infektion, såsom produktion af antimikrobielle peptider og andre effektormolekyler 4, 5. Hemocyte morfologi og funktion er hovedsageligt blevet undersøgt i genetiske eller fysiologiske insekt modeller, herunder bananfluen, Drosophila melanogaster 6, 7, myggene Aedes aegypti og Anopheles gambiae 8, 9 og tobak hornworm, Manduca sexta 10, 11. Imidlertid kun få oplysninger i øjeblikket findes om den mangfoldighed, klassificering, morfologi og funktion hæmocytter i ikke-model insektarter, især dem indsamlet i naturen 12. Her beskriver vi en enkel og effektiv protokol til ekstraktion hæmocytter fra vilde larver. Vi bruger næstsidste stadium Lithacodes Fasciola (gul-bredskuldret slug møl) (figur 1) og Euclea delphinii (spiny eg slug) larver (Lepidoptera: Limacodidae), og vise at der er tilstrækkelige mængder af hemolymph (insekt blod)kan isoleres og hemocyte numre talt fra individuel larver. Denne fremgangsmåde kan anvendes til effektivt at undersøge hemocyte typer i disse arter såvel som i andre beslægtede skadelige sommerfugle larver høstet fra marken, eller den kan let kombineres med immunologiske assays til formål at undersøge hemocyte funktion efter infektion med mikrobielle eller parasitiske organismer 13.

Protokollen er beskrevet heri mulighed for indsamling af et minimum volumen på 10-20 ul insekt hemolymph fra de enkelte larver. Hæmocytter indsamlet ved denne metode er fri for celle-sammenklumpning melanin defekter, vævsrester eller andre forureninger. Derfor hæmocytter let kan observeres og talt under mikroskop, og flere insekter kan observeres indenfor få Hosur. Vi observerede, at de fleste hæmocytter i vores prøver bestod af plasmatocytes 3 ​​(hæmocytter spredes 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 hæmocytter udfører mange vigtige funktioner, både immune og ikke-immune, i alle faser af insekt udvikling. Vores nuværende viden om hemocyte typer og funktion kommer fra undersøgelser af insekt genetiske modeller. Her præsenteres en metode til udvinding, kvantificere og visualisere hæmocytter fra vilde sommerfuglelarver.

En enkel protokol til Udpakning hæmocytter fra Wild Larver

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

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