Gli autori sono in debito con i capi tribù, capi e le comunità di Macha per la loro collaborazione nel corso unwaveing ​​collezioni zanzara campione sul campo. Il dottor Doug e Rebecca Norris Kent offerto competenze di alta qualità sul protocollo salatura fuori. Questo lavoro è stato finanziato dalla Johns Hopkins Malaria Research Institute sistema pilota di sovvenzione. Mosquito e parassiti del DNA standard di laboratorio sono stati gentilmente donati da MR4, tipo American &amp; Culture Collection.

1. Dissezione di preparazione dei campioni Mosquito <ol><li> Zanzara Posto su un piccolo taglio di parafilm e supporto sulla dissezione microscopio. </li><li> Copertina in goccia di acqua deionizzata per ammorbidire i tessuti. </li><li> Incidere la carcassa zanzara proprio in corrispondenza della giunzione tra il torace e l&#39;addome, in modo da intaccare la testa e il torace dalla sezione addominale. </li><li> Trasferire ogni sezione zanzara sezionato in un ambiente pulito in autoclave 1,5 ml tubetto microcentrifuga prelabeled con dettagli zanzare numero ID e che portano l&#39;etichetta per indicare la sezione del corpo (ad esempio, &quot;m&quot; per la metà-gut sezione, &quot;ht&quot; per testa e torace sezione). </li><li> Eliminare parafilm e asciugare con cura tavolino del microscopio con il 70% di alcool-imbevuto Kimwipe prima di elaborare la zanzara successivo. </li></ol> 2. Estrazione del DNA da campioni Mosquito <ol><li> Pipettare 20 ml di acqua deionizzata in provetta e l&#39;uso della punta di pipetta per macinare la sezione zanzara sommerso in una ONUsospensione iForm. </li><li> Aggiungere 100 ml di PBS 1X autoclavato / soluzione di 1% di saponina omogenato del campione e mescolare dolce vortex momentanea. </li><li> Incubare a temperatura ambiente per 20 min. </li><li> Centrifugare a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Risospendere il pellet in 100 ul di PBS 1X. </li><li> Centrifugare nuovamente a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Vortexando gentilmente (5 sec), risospendere il pellet in 75 microlitri di acqua sterile deionizzata e 25 ml di 20% w / v Chelex-100 sospensione resina in acqua deionizzata. </li><li> Pierce foro nel coperchio della provetta sterile con ago ipodermico 23G fiammato in becco Bunsen. </li><li> Ebollizione sospensione del campione in bagnomaria a cremagliera galleggiante (o in blocco di riscaldamento) per 10 min. </li><li> Centrifugare a 20.000 xg per 1 min e soluzione di DNA conseguente trasferimento nella fiala stoccaggio prelabeled per uso come modello in applicazioni PCR. </li></ol> 3. Plasmodium falciparum Genotipizzazione e Anophelesspp. Identificazione molecolare <ol><li> Aggiungere 2,5 ml di estratto DNA Chelex in 25 ul reazioni PCR per verificare la qualità del DNA 11, identificare le specie Anopheles 10 e al genotipo P. falciparum 12 infezioni della ghiandola metà del budello e salivari. </li><li> Per massimizzare la resa amplicone, soprattutto per P. rilevamento falciparum, includono 1.5X albumina di siero bovino (BSA) in PCR. La composizione seguente reazione è raccomandato: (2,5 template pl, 0,25 primers pm, 1,5 mM di cloruro di magnesio, 200 microM dNTPs, 1X PCR Buffer, 1.5X BSA e 1.0U Taq DNA polimerasi, in 25 volumi pl). </li><li> Per verificare la qualità del DNA in estratto, amplificano regione del artropodi deidrogenasi mitocondriale nicotinammide adenin dinucleotide (NADH) subunità 4 (ND4) gene (primer ND4FW [5&#39;-GTD YAT TTA TGA TTR CCT ​​AA-3 &#39;] e ND4RV [5&#39; CTT-CGD CTT CCW ADW CGT TC-3 &#39;]; aspettiamo prodotto 400BP) 11,13. </li><li> Per differenziare Anopheles gambiae SL. specie sorella (An. gambiae ss e An. arabiensis solo), amplificare affiancano la regione SNPs nel distanziale intergenica (IGS) regione (primer ONU [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; aspettiamo 390bp prodotto una SS gambiae, 315bp arabiensis An) 10,11. </li><li> Per la tipizzazione P. falciparum antifolato polimorfismi di resistenza ai farmaci, eseguire nested PCR per amplificare regione fiancheggiante codoni amminoacidici 108, 51, 59, 16 e 164 nella parassita diidrofolato reduttasi (DHFR) gene: Primer rotonde primari: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] e M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] Secondari primer rotonde: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] con F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] O F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] con M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). Anche amplificare regione contenente codoni amminoacidici 436, 437, 540, 581 And 613 nel P. falciparum diidropteroato sintetasi (DHPS) gene: Primaria tutto primer R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] con R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; Secondari primer rotonde J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] con K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] O K: [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;] con K /, O L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] con L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Soggetto 4 amplicone microlitri di digestioni di restrizione allele-specifici enzimi in 30 ul reazioni seguendo le istruzioni del fabbricante, di rilevare antifolato farmaci associate a resistenza polimorfismi 12. </li><li> Soggetto 5 pl di ampliconi PCR (o 15 ml di digestione di restrizione) per pista di bromuro di etidio-colorato gel di agarosio al 2% per elettroforesi (100 - 120V) e visualizzare le bande con transilluminazione UV. </li></ol>

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Nessun conflitto di interessi dichiarati.

Il metodo semplificato Chelex qui presentato permette l&#39;estrazione di qualità Anopheles spp e P. falciparum DNA da campioni di zanzara suscettibili di diverse applicazioni PCR. Questa tecnica può essere utilizzata per l&#39;identificazione molecolare di zanzare vettore malaria e la sorveglianza della farmaco-resistenti P. alleli falciparum in zanzare per i programmi nazionali di controllo della malaria. I vantaggi della tecnica Chelex semplificata includono semplicità, meno reattivi e quindi dei costi, sicurezza e tempi di lavorazione più brevi (37 min) di protocolli standard quali il salting out metodo 10 (2 h 47 min e un passo overnight, Tabella 1). I vantaggi di cui sopra ed i requisiti minimi dei reagenti (3 reagenti) rispetto al protocollo standard attuale (10 reagenti, Tabella 1) 11,15, rendono il protocollo Chelex semplificato particolarmente cordiale ai laboratori dei paesi endemici in cui un reagente costantefiliera è spesso difficile da mantenere. Una limitazione del metodo è che, come il protocollo standard, è stato anche oggetto di inibitori della PCR noti a verificarsi nel tegumento zanzara 16. Tuttavia, questo è facilmente alleviato inclusione di BSA nei saggi. BSA è stata utilizzata con successo come amplificazione enhancer contro inibitori in altre applicazioni PCR 17,18.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Chelex-100, 50-100 mesh a secco </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> Saponina </td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> New England Biolabs </td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

Paesi endemici stanno sempre più adottando strumenti molecolari per la tipizzazione efficiente, identificazione e sorveglianza contro i parassiti della malaria e le zanzare vettore, come parte integrante dei loro programmi di controllo 1,2,3,4,5. Per la creazione sostenibile di questi approcci precisi nelle operazioni di ricerca per rafforzare il controllo della malaria e gli sforzi di eliminazione, metodi semplici e convenienti, con reagente parsimoniosa e alle attrezzature sono essenziali 6,7,8. Qui vi presentiamo una semplice Chelex tecnica basata per l&#39;estrazione parassita della malaria e il DNA da campioni di zanzara vettore raccolti sul campo. Abbiamo identificato morfologicamente 72 Anopheles gambiae sl. Da 156 zanzare catturate dalla piretro spruzzo catture accessorie nelle camere da letto delle famiglie all&#39;interno di un km 2.000 2 pressi dell&#39;Istituto malaria a Macha. Dopo la dissezione, per separare la testa e il torace dall&#39;addome per tutti Anopheles 72 gambiae sl. zanzare, le due sezioni sono state singolarmente messo in tubi microcentrifuga da 1,5 ml e immerso in 20 ml di acqua deionizzata. Usando una pipetta sterile, ogni sezione zanzara stato omogeneizzato separatamente ad una sospensione uniforme in acqua deionizzata. Dell&#39;omogenato derivanti da ciascuna sezione zanzara, 10 microlitri è stata conservata mentre l&#39;altro 10 microlitri è stato trasferito ad una provetta separata autoclavata 1,5 ml. Le aliquote separate sono stati sottoposti ad estrazione del DNA sia dal Chelex semplificata o lo standard salting out estrazione protocollo 9,10. Il protocollo salting out è il cosiddetto e ampiamente utilizzati perché impiega elevate concentrazioni saline in luogo di pericolosi solventi organici (come fenolo e cloroformio) per la fase di precipitazione della proteina durante l&#39;estrazione del DNA 9. Gli estratti sono stati utilizzati come modelli per l&#39;amplificazione PCR utilizzando primers mirati artropodi mitocondriale dehydroge nicotinammide-adenina-dinucleotidenase (NADH) subunità 4 gene (ND4) per verificare la qualità del DNA 11, una PCR per l&#39;identificazione di specie Anopheles gambiae fratelli 10 e un nested PCR per la tipizzazione di infezione da Plasmodium falciparum 12. Confronto con qualità del DNA (ND4) PCR ha mostrato il 93% di sensibilità e specificità 82% per l&#39;approccio Chelex relativo alla stabilita salatura fuori protocollo. Corrispondenti valori di sensibilità e specificità sono state del 100% e 78%, rispettivamente, con fratello identificazione delle specie PCR e il 92% e 80%, rispettivamente, per P. falciparum rilevamento PCR. Non ci sono state differenze significative nella percentuale di campioni che con il segnale amplificato Chelex o il regolare salatura su protocollo in tutte e tre le applicazioni PCR. L&#39;approccio Chelex richiesto tre reagenti semplici e 37 min per completare, mentre la salatura su protocollo ha comportato 10 diversi reagenti e 2 ore e 47 min il tempo di elaborazione &#39;, tra cui un passo durante la notte. I nostri risultati mostrano °al metodo Chelex è paragonabile a quella esistente salting out estrazione e può essere sostituito da un approccio semplice e sostenibile in risorse limitate, dove una costante filiera reagente è spesso difficile da mantenere.

Esempi di risultati di saggi di PCR per la qualità zanzara estratto DNA (Figura 1), arabiensis Anopheles identificazione molecolare (Figura 2) e P. falciparum rilevamento (Figura 3) mostra che il metodo semplificato Chelex produce risultati simili allo standard salting out protocollo 10, nonostante passi molto minori (Tabella 1). Con la qualità del DNA comparabile nei rispettivi estratti non è sorprendente che la positività frequenze di campionamento per quanto riguarda An. specie sorella gambiae così come i tassi di infezione del parassita non erano statisticamente differenti in base a McNemar test Chi-quadrato (Figura 3). Sensibilità (%) è stata calcolata come VP / (VP + FN) * 100, dove TP denota veri positivi e falsi negativi FN indica. Specificità (%) è stato determinato come TN / (TN + FP) * 100, dove TN denota veri negativi e FP denota falsi positivi. La qualità del DNA (ND4) PCR ha mostrato il 93% di sensibilità e specificità 82% per l&#39;approccio Chelex rispetto alla norma stabilita salatura fuori protocollo come l&#39;oro. Corrispondenti valori di sensibilità e specificità sono state del 100% e 78%, rispettivamente, con fratello identificazione delle specie PCR e il 92% e 80%, rispettivamente, per P. falciparum rilevamento PCR. L&#39;aggiunta di BSA a miscele di reazione determinato un generale aumento di PCR positivi (Figura 3) dovute ai rilievi inibitori della PCR 14, sia per il Chelex semplificato e regolare salting out protocollo. Tuttavia, questo aumento non era statisticamente significativo tranne che per qualità del DNA (ND4 PCR) su estratti Chelex (p = 0.039). Allele-specifica digestione con enzimi di restrizione P. falciparum DHFR (o altro gene target) amplicon consente la genotipizzazione di infezioni metà ghiandola salivare intestinale e malaria per alleli di resistenza ai farmaci (Figura 4; ghiandole salivari dati non mostrati). <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Chelex procedura semplice </td><td colspan="2"> Standard salatura fuori procedura </td></tr><tr><td> Passi </td><td> Reagenti </td><td> Passi </td><td> Reagenti </td></tr><tr><td><ol><li> Aggiungere al campione provetta 1,5 ml microcentrifuga e omogeneizzare in 100 pl di PBS 1X / saponina soluzione 1% (2 min). </li><li> Lascia a temperatura ambiente per 20 min. </li><li> Spin a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Aggiungere 100 ml di PBS 1X e miscelare delicatamente momentaneamente vortex (3 sec). </li><li> Spin a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Aggiungere 25 ml di 20% w / v Chelex in acqua deionizzata e 75 microlitri di acqua sterile deionizzata. </li><li> Pierce foro nel coperchio del tubo campione utilizzando caldo, ago ipodermico sterile 23G e contenuto della provetta bollire per 10 min. </li><li> Spin provetta per microcentrifuga a 20.000 xg per 1 min. </li><li> Trasferire il surnatante in fiala sterile prelabeled e DNA soluzione conservare a -20 ° C fino al momento dell&#39;uso (2,5 microlitri in 25 microlitri di reazione PCR). </li></ol></td><td> PBS Saponina Chelex-100 perle </td><td><ol><li> Aggiungere al campione provetta 1,5 ml microcentrifuga e omogeneizzare in 100 microlitri tampone * Bender con 1% DEPC (2 min). </li><li> Incubare a 65 ° C per 1 ora. </li><li> Aggiungere 15 microlitri fredda 8 M acetato di potassio. Mescolare delicatamente e incubare in ghiaccio per 45 min. </li><li> Spin campione in provette per microcentrifuga a 20.000 xg per 10 min e il trasferimento supernatante in una nuova provetta 1,5 ml. </li><li> Aggiungere 250 microlitri di etanolo al 100% e mescolare bene capovolgendo la provetta. </li><li> Incubare il campione a temperatura ambiente per 5 min. </li><li> Centrifugare i campioni a 20.000 xg per 15 min. </li><li> Aspirare ed eliminare il surnatante, il pellet lasciando asciugare completamente nel tubo (30 ma). </li><li> Risospendere il pellet in 20 ml di 0,1 X. SSC + RNAsi (10 mg / ml) per una notte a 4 ° C. </li><li> Aggiungere 80 microlitri di acqua DEPC e conservare a -20 ° C fino al momento dell&#39;uso. </li></ol></td><td> Dietilpirocarbonato (DEPC) * Bender Buffer: 0,1 M NaCl 0,2 M Saccarosio 0,1 M Tris-HCL 0,05 M EDTA, pH 9,1 0,5% SDS in acqua DEPC 8 M di acetato di potassio Etanolo 0,1 X. Saline di sodio citrato (SSC) tampone RNAsi (10 mg / ml) </td></tr><tr><td colspan="2"> TEMPO TOTALE: 37 min </td><td colspan="2"> TEMPO TOTALE: 2 h 47 min, più durante la notte </td></tr></tbody></table> Tabella 1. Confronto passo per passo dei reagenti e dei tempi necessari per il protocollo Chelex semplice e standard salatura metodo di estrazione del DNA da campioni di zanzara. <img alt="Figura 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Figura 1. ND4 mitocondriale confronto PCR di DNA di qualità semplificato Chelex &quot;C&quot; e standard salting out &quot;M&quot; su campioni estratti arabiensis campo Anopheles. NC, controllo negativo, M1 e C1, C2 e M2, M3 e C3, C4 e M4 e sono accoppiati salatura ed estratti Chelex da An stessa. arabiensis zanzara campioni. L, 100 bp DNA ladder. <img alt="Figura 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> Figura 2 molecolare PCR per l&#39;identificazione arabiensis Anopheles C1 e M1, M2 e C2, C3 e M3, M4 C4 e denotano rispettivamente da amplicon salting out &quot;M&quot; e semplificato Chelex &quot;C&quot; estratti di DNA per gli esemplari di zanzara stessi,.. AR, Anopheles arabiensiscontrollo positivo, L, 100 bp DNA ladder. <img alt="Figura 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Figura 3. Rilevamento di positivi su Chelex e salatura su DNA estratto da campioni prelevati sul campo di zanzara, con saggi di PCR per l&#39;identificazione molecolare di Anopheles arabiensis (AR) 10, artropodi gene NADH deidrogenasi 4 (ND4) qualità del DNA test 11, e antifolato P resistenza ai farmaci . falciparum DHFR genotipizzazione 12 (F-M4). Risultati visualizzati per dosaggi con o senza BSA nella miscela di reazione. McNemar chi-square test è stato utilizzato per determinare se le differenze tra cento di PCR positivi da DNA estratto dal Chelex semplificata e lo standard salting out protocolli erano statisticamente significative. <img alt="Figura 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Figura 4. Applicazione del protocollo semplificato Chelex genotipizzazione P. infezioni falciparum in zanzare (mid-gut dati illustrati) per gli alleli di resistenza ai farmaci. BstN ho la digestione su amplicone accompagnamento codone 108 del P. falciparum gene DHFR 12 mostra cicloguanile mutanti resistenti S108T in campioni di zanzara (M1-M5; metà gut dati riportati). U, non digerito 522 bp ampliconi, FCR3, standard di laboratorio P. falciparum clone controllo positivo svolgimento S108T, K1, P. falciparum standard di laboratorio di controllo clone negativo portando cicloguanile sensibile S108N, L, 100 bp DNA ladder.

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

Un semplice protocollo Chelex per l'estrazione del DNA da Anopheles spp. | Immunologia e infezione | Video

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Un modo rapido e conveniente per estrarre parassita della malaria qualità e il DNA da campioni di zanzara vettore è descritto. Capitalizzando proprietà chelanti di resina Chelex, il semplice metodo consente genotipizzazione dei parassiti della malaria in fasi zanzara ghiandole mid-intestinali e salivare, nonché identificazione molecolare del<em&gt; Anopheles</em&gt; Sibling specie mediante PCR.

Un protocollo Chelex semplice per l&#39;estrazione del DNA da<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.8K Views.  Malaria Institute at Macha. Un modo rapido e conveniente per estrarre parassita della malaria qualità e il DNA da campioni di zanzara vettore è descritto. Capitalizzando proprietà chelanti di resina Chelex, il semplice metodo consente genotipizzazione dei parassiti della malaria in fasi zanzara ghiandole mid-intestinali e salivare, nonché identificazione molecolare del Anopheles Sibling specie mediante PCR.

Video: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Immunologia e infezioni

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

Tecnologia IgY: Estrazione di anticorpi pollo rosso d&#39;uovo con polietilenglicole (PEG) Precipitazioni

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Schermi throughput elevato e basso con Nematodi galligeni Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Un semplice protocollo per Clumping piastrinica mediata da<em&gt; Plasmodium falciparum</emEritrociti&gt; infette in una risorsa Povero Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per<em&gt; Burkholderia</em&gt; Specie

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocollo per l&#39;analisi dell&#39;epatite C replicazione del virus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l&#39;analisi di linfociti infiltranti

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Un protocollo Chelex semplice per l'estrazione del DNA da

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla Aedes aegypti

Tecnologia IgY: Estrazione di anticorpi pollo rosso d'uovo con polietilenglicole (PEG) Precipitazioni

Schermi throughput elevato e basso con Nematodi galligeni Meloidogyne spp.

Un semplice protocollo per Clumping piastrinica mediata da

Totale Estrazione di proteine e 2-D elettroforesi su gel Metodi per

Un protocollo per l'analisi dell'epatite C replicazione del virus

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l'analisi di linfociti infiltranti

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

Un protocollo Chelex semplice per l'estrazione del DNA da

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla Aedes aegypti

Tecnologia IgY: Estrazione di anticorpi pollo rosso d'uovo con polietilenglicole (PEG) Precipitazioni

Schermi throughput elevato e basso con Nematodi galligeni Meloidogyne spp.

Un semplice protocollo per Clumping piastrinica mediata da

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per

Un protocollo per l'analisi dell'epatite C replicazione del virus

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l'analisi di linfociti infiltranti

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

Totale Estrazione di proteine e 2-D elettroforesi su gel Metodi per