Nous tenons à remercier Sean Fitzwater et Carmen Luna Giannina de Colombo pour le segment de croissance de tuberculose vidéo time-lapse. Nous tenons à remercier Marty Roper pour sa rétroaction approfondie et un excellent cours de l&#39;édition et co-auteur du Guide de l&#39;utilisateur, à partir de laquelle le protocole actuel a été prise essentiellement verbatim. La production de cette vidéo a été financée par le NIH / Fogarty International Center <a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a> David Moore AJ contribué en tant que boursier de recherche clinique de Wellcome Trust en Médecine Tropicale et Reader en maladies infectieuses de l&#39;Imperial College de Londres (Fellowship 078067/Z/05 plusieurs prix). Mark F. Brady contribué tant que boursier du NIH / Fogarty International Research Center.

<ol><li> Préparer des solutions étalons <ol><li> Stock tampon phosphate <ol><li> Mélanger 950ml de sodium dibasique solution (9.47g de phosphate dibasique de sodium dissous dans l&#39;eau distillée 1000ml) avec 950ml de solution de phosphate de potassium monobasique (9.07g de phosphate de potassium monobasique dissous dans l&#39;eau distillée 1000ml) et remuer; retenir 50ml de chaque solution pour ajuster pH si nécessaire </li><li> Ajuster le pH à 6,8 ± 0,2: ajouter le phosphate de sodium dibasique solution pour augmenter le pH, ajouter une solution de phosphate de potassium monobasique pour abaisser le pH </li><li> Autoclave à 121-124 ° C pendant 15 minutes pour stériliser </li><li> Assiette une aliquote de 100 ul de la solution tampon phosphate sur milieu nutritif gélosé dans une boîte de Pétri et incuber à 37 ° C pendant 48 heures pour confirmer la stérilité </li><li> Conserver au réfrigérateur à 2-8 ° C pendant jusqu&#39;à un mois Remarque: Chaque échantillon d&#39;expectoration nécessite 10ml de solution tampon phosphate </li></ol></li><li> Décontamination des stocks <ol><li> Combiner des volumes égaux de solution d&#39;hydroxyde de sodium (8.0g d&#39;hydroxyde de sodium dissous dans 200ml d&#39;eau distillée stérile) et la solution de citrate de sodium (5.8g de citrate de sodium distillée dans 200ml d&#39;eau distillée stérile) </li><li> Mélanger et autoclave à 121-124 ° C pendant 15 minutes </li><li> Conserver au réfrigérateur à 2-8 ° C pendant jusqu&#39;à un mois Remarque: Chaque échantillon d&#39;expectoration nécessite 2ml de stock de décontamination </li></ol></li><li> Milieux de culture des stocks <ol><li> Into 900ml d&#39;eau distillée stérile ajouter 3.1ml de glycérol et 1,25 g d&#39;casitone et 5,9 g de poudre de support 7H9; mélanger avec une agitation constante jusqu&#39;à dissolution complète </li><li> Autoclave à 121-124 ° C pendant 15 minutes </li><li> Laisser refroidir et diviser le milieu stérile en aliquots de 4,5 ml de stériles 16x 100 mm pour des tubes de verre de préparation d&#39;échantillon, et 10.8ml aliquotes pour la préparation des solutions antibiotiques et des contrôles internes </li><li> Incuber à 37 ° C pendant 48 heures pour vérifier la stérilité (absence de turbidité) </li><li> Conserver à 2-8 ° C avec bouchon fermé pour jusqu&#39;à un mois Remarque: Chaque échantillon d&#39;expectoration nécessite 4,5 ml d&#39;un tube contenant du milieu 7H9 </li></ol></li><li> Solutions concentrées d&#39;antibiotiques <ol><li> A. isoniazide stock (8 mg / ml) <ol><li> Dissoudre 20 mg d&#39;isoniazide complètement dans 2.5ml d&#39;eau distillée stérile </li><li> Filtre avec filtre seringue 0,2 um pour le solvant aqueux </li><li> Boutique en aliquots de 20 pi stériles tubes de centrifugation micro à -20 ° C pendant jusqu&#39;à 6 mois Remarque: Chaque aliquote stocké 20 pi est suffisant pour un maximum de 100 échantillons (y compris les pertes) </li></ol></li><li> B. La rifampicine stock (8 mg / ml) </li></ol><ol><li> Dissoudre la rifampicine 20mg complètement dans 1,25 ml de DMSO </li><li> Ajouter 1,25 ml d&#39;eau distillée stérile et mélanger </li><li> Filtre avec filtre seringue 0,2 um pour les solvants organiques </li><li> Boutique en aliquots de 20 pi microtubes stériles à -20 ° C pendant jusqu&#39;à 6 mois Remarque: Chaque aliquote stocké 20 pi est suffisant pour un maximum de 100 échantillons (y compris les pertes) </li></ol></li></ol></li><li> Préparer des solutions de travail <ol><li> Solution de décontamination de travail <ol><li> Dissoudre 0,1 g de cristaux NALC dans toutes les 20ml de solution de décontamination requis Remarque: Chaque échantillon doit de 2ml de solution de décontamination Remarque: Jeter toute solution de décontamination NaOH NALC qui reste inutilisée après 24 heures que NALC perd son activité mucolytique au fil du temps </li></ol></li><li> Milieux de culture solution de travail <ol><li> Partez: <ol><li> 1 tube avec support 7H9 pour chaque échantillon d&#39;expectoration à traiter, plus 1 tube supplémentaire pour chaque plaque (pour la colonne de contrôle négatif) </li><li> 2 tubes de milieu 7H9 pour les contrôles positifs (3 si des souches monorésistants sont utilisés) </li><li> 1-2 tubes avec 7H9 10.8ml pour la préparation de solution d&#39;antibiotiques </li></ol></li><li> Ajouter 0,5 ml OADC à l&#39;4,5 ml d&#39;7H9 dans chaque tube d&#39;échantillon </li><li> Ajouter 1,2 ml d&#39;OADC tubes avec 7H9 10.8ml </li><li> Mettez de côté deux tubes avec 5ml 7H9-OADC pour les contrôles positifs, et le tube (s) avec 12ml 7H9-OADC être utilisé pour la préparation solution antibiotique (ceux-ci ne nécessitent PANTA) </li><li> PANTA Reconstituer et ajouter 0,1 ml dans chaque tube d&#39;échantillon et les tubes de contrôle négatif (7H9-OADC-PANTA: volume total = 5.1ml) Remarque: milieu complet avec PANTA (7H9-OADC-PANTA) est utilisée pour les échantillons de crachats et les contrôles négatifs, l&#39;utilisation 7H9-OADC sans PANTA pour les contrôles positifs et pour la préparation de solution d&#39;antibiotiques </li></ol></li><li> Antibiotique solutions de travail <ol><li> 1. Voir la Figure 1 pour les étapes à faire de la solution antibiotique travaillant dans une partie inutilisée de 24 puits Plaque Note: Pour les résultats de sensibilité précis, les concentrations finales des antibiotiques sont essentiels. La procédure suivante est adapté pour lABORATOIRES équipé micropipettes. Si micropipettes sont pas disponibles, des seringues à tuberculine peut être utilisé avec une autre série de dilutions décrites dans le Guide de l&#39;utilisateur Annexe 3 au modsperu.org Note: Ne pas recongeler ou réutilisation de travail des antibiotiques ou des solutions de stock que l&#39;activité des médicaments peuvent être perdus Ecarter les solutions inutilisées aux antibiotiques à la fin de la figure 1 jour de traitement.. Rendre les solutions antibiotiques de travail (de Guide de l&#39;utilisateur au modsperu.org) </li></ol></li></ol></li><li> Échantillon de crachats et de la plaque préparation <ol><li> De décontamination <ol><li> 2ml Lieu de crachats dans un tube à centrifuger 15ml (si moins, faire jusqu&#39;à 2ml avec un tampon phosphate; si plus, utilisez uniquement 2ml) </li><li> Ajouter 2 ml de NaOH NALC solution </li><li> Tube de Cap hermétiquement et vortex pendant 20 secondes; Retourner le tube d&#39;assurer des contacts solution de NaOH-NALC toute la surface intérieure du tube et le couvercle </li><li> Laisser reposer pendant un minimum de 15 minutes - peut prolonger de quelques minutes si l&#39;échantillon est particulièrement visqueux. Pour éviter plus de traitement, ne doivent pas dépasser 20 minutes </li><li> Remplir le tube d&#39;14ml avec un tampon phosphate (pH 6,8) pour neutraliser les alcalis et terminer le processus de décontamination, et bien mélanger en inversant le tube 4 fois </li><li> Centrifuger à 3000 g pendant 15 minutes (voir l&#39;annexe 2 du Guide de l&#39;utilisateur au modsperu.org pour l&#39;équation de rotations par minute nécessaire pour atteindre 3000 g) </li><li> Versez délicatement le surnageant dans un récipient de déchets liquides à l&#39;hypochlorite de sodium à 10% ou un autre désinfectant approprié et conserver le culot </li></ol></li><li> Préparation de la suspension de l&#39;échantillon final et de sauvegarde <ol><li> Utiliser 7H9-OADC-PANTA (à partir du tube contenant 5.1ml), resuspendre le culot de l&#39;échantillon dans un volume total de 2ml dans le tube de centrifugeuse avec une pipette Pasteur et bien mélanger </li><li> Retirer 1ml de la suspension de l&#39;échantillon et de stocker dans un microtube à 2-8 ° C en tant que sauvegarde </li><li> Ajouter la seconde 1ml de la suspension de l&#39;échantillon dans le tube avec le reste 7H9-OADC-PANTA et bien mélanger. C&#39;est la solution de l&#39;échantillon final soit prêt pour le placage </li></ol></li><li> Placer l&#39;échantillon dans les suspensions de 24 puits Plaque <ol><li> 900μl lieu de la suspension de l&#39;échantillon final dans chacun des 4 puits d&#39;une colonne dans la plaque de 24 puits </li><li> Répétez l&#39;opération avec d&#39;autres échantillons jusqu&#39;à ce que toutes les colonnes de la plaque, sauf la colonne 3, sont remplies (ou jusqu&#39;à ce que tous les échantillons sont plaqués) </li><li> Lieu de 900μl 7H9-OADC milieu sans échantillon dans le puits 4 de la colonne 3 de chaque plaque échantillon (négatif contrôles internes) </li></ol></li><li> Ajout d&#39;antibiotiques à des échantillons <ol><li> Avec une pipette multi-canaux, remplir soigneusement quatre pointes avec 100 ul dans les puits avec 7H9-OADC et antibiotique solutions de travail (colonne 2 de la dilution des antibiotiques plaques voir figure 1) </li><li> Ajouter les aliquotes 100 ul de la colonne 1 puits avec des solutions d&#39;échantillon 900μl sans toucher la plaque ou un échantillon </li><li> Répétez jusqu&#39;à ce que toutes les colonnes ont reçu les 100 ul supplémentaires de moyenne (sans drogue puits) ou des solutions antibiotiques (y compris les colonnes de contrôle négatif 3) </li><li> Refermez la plaque avec son couvercle et placer dans un refermable en polyéthylène (Ziplock) sac et sceller (sac n&#39;est pas ouvert à nouveau à partir de ce point) </li><li> Incuber à 37 ° C (enrichissement en CO 2 n&#39;est pas nécessaire) </li></ol></li></ol></li><li> Contrôle de la qualité <ol><li> Des contrôles négatifs sont exécutés sur chaque assiette. La présence de toute la croissance dans l&#39;un des puits dans la colonne de contrôle négatif indique une contamination croisée, par conséquent toute la plaque doit être mis au rebut et les échantillons re-plaqué. </li><li> Les contrôles positifs sont exécutés sur une plaque séparée, à la fin de la journée. Un totalement sensibles aux médicaments souche est exécuté dans une colonne et une souche résistante à l&#39;INH et RIF est exécuté dans une autre colonne. Si on se préoccupe de l&#39;exécution d&#39;une souche multirésistante, à la fois une contrainte et une INH monorésistants FRR souche monorésistants peut être exécuté à l&#39;endroit des souches sensibles et MDR. Tous les contrôles positifs doivent avoir une croissance dans le puits sans médicament pour démontrer que le soutien à la croissance des médias. Pour démontrer que les concentrations de médicament sont corrects, la souche sensible ne devraient pas croître dans l&#39;INH ou FRR puits, mais la souche multirésistante doit croître aussi se développer dans l&#39;INH et RIF puits. </li></ol></li><li> Lecture des plaques <ol><li> Avec le jour de plaquage comme «Jour 0&quot;, la lecture de la plaque commence le jour 5, et si elle est négative le jour 5 lecture se fait chaque jour (ou deux jours selon la charge de travail) jusqu&#39;au jour 14, puis tous les 2-3 jours à partir de 15 jours -21. Les plaques sont examinées sur unemicroscope inversé la lumière avec l&#39;objectif 10x </li><li> Une culture positive est celle dans laquelle il ya deux ou plusieurs unités formant colonies (&gt; 2 UFC) dans les deux puits sans drogue. Au début de croissance mycobactérienne ressemble petites virgules courbes ou spirales (jours 5-9). La formation de colonies progresse habituellement de «cordes» ou «enchevêtrements» (voir la bibliothèque d&#39;images sur le modsperu.org) </li><li> Le jour d&#39;une culture est positive dans les deux puits sans drogue, les puits contenant un médicament doit être lu. Toute la croissance de la présence d&#39;un médicament indique la résistance à ce médicament. Remarque: Si la lecture des puits contenant des médicaments se fait&gt; 1 semaine après une culture positive est noté dans le puits sans drogue, cela peut ne pas représenter la résistance que la percée de croissance est parfois observée </li><li> Les échantillons qui ne sont pas positifs au jour 21 sont considérées comme des cultures négatives </li><li> La nébulosité des puits est le résultat d&#39;une prolifération de contamination, alors que la croissance mycobactérienne ne résulte pas de la nébulosité </li><li> Au terme de 21 jours des cultures peut être jeté. Cultures rester dans leurs sacs scellés et sont placés dans des sacs en autoclave et autoclave à 121-124 ° C pendant 45-60 minutes </li></ol></li></ol>

<ol>
	<li>Arias, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic-observation+drug-susceptibility+assay+for+detection+of+tuberculosis.">Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis.</a> Clin Infect Dis. 44, 674-674 (2007).</li><li>Caviedes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+efficient+detection+and+drug+susceptibility+testing+of+Mycobacterium+tuberculosis+in+sputum+by+microscopic+observation+of+broth+cultures.+The+Tuberculosis+Working+Group+in+Peru.">Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru.</a> J Clin Microbiol. 38, 1203-1203 (2000).</li><li>Caviedes, L., Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+MODS:+a+low-cost,+low-tech+tool+for+high-performance+detection+of+tuberculosis+and+multidrug+resistant+tuberculosis.">Introducing MODS: a low-cost, low-tech tool for high-performance detection of tuberculosis and multidrug resistant tuberculosis.</a> Indian J Med Microbiol. 25, 87-87 (2007).</li><li>Caws, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+MODS+culture+technique+for+the+diagnosis+of+tuberculous+meningitis.">Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis.</a> PLoS ONE. 2, e1173-e1173 (2007).</li><li>Ejigu, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic-observation+drug+susceptibility+assay+provides+rapid+and+reliable+identification+of+MDR-TB.">Microscopic-observation drug susceptibility assay provides rapid and reliable identification of MDR-TB.</a> Int J Tuberc Lung Dis. 12, 332-332 (2008).</li><li>Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+occupational+tuberculosis+in+National+Tuberculosis+Programme+laboratories+in+Korea.">Risk of occupational tuberculosis in National Tuberculosis Programme laboratories in Korea.</a> Int J Tuberc Lung Dis. 11, 138-138 (2007).</li><li>Mello, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic+observation+drug+susceptibility+assay+for+detection+of+Mycobacterium+tuberculosis+resistance+to+isoniazid+or+rifampin.">Clinical evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to isoniazid or rifampin.</a> J Clin Microbiol. 45, 3387-3387 (2007).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+prospects+for+the+MODS+assay+in+multidrug-resistant+tuberculosis+diagnosis.">Future prospects for the MODS assay in multidrug-resistant tuberculosis diagnosis.</a> Future Microbiol. 2, 97-97 (2007).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrequent+MODS+TB+culture+cross-contamination+in+a+high-burden+resource-poor+setting.">Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting.</a> Diagn Microbiol Infect Dis. 56, 35-35 (2006).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic-observation+drug-susceptibility+assay+for+the+diagnosis+of+TB.">Microscopic-observation drug-susceptibility assay for the diagnosis of TB.</a> N Engl J Med. 355, 1539-1539 (2006).</li><li>Moore, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+observation+drug+susceptibility+assay,+a+rapid,+reliable+diagnostic+test+for+multidrug-resistant+tuberculosis+suitable+for+use+in+resource-poor+settings.">Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings.</a> J Clin Microbiol. 42, 4432-4432 (2004).</li><li>Moore, D. A., Roper, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+smear-negative+tuberculosis+in+people+with+HIV/AIDS.">Diagnosis of smear-negative tuberculosis in people with HIV/AIDS.</a> Lancet. 370, 1033-1033 (2007).</li><li>Oberhelman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+recovery+of+Mycobacterium+tuberculosis+from+children+using+the+microscopic+observation+drug+susceptibility+method.">Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method.</a> Pediatrics. 118, e100-e100 (2006).</li><li>Palomino, J. C., Martin, A., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MODS+assay+for+the+diagnosis+of+TB.">MODS assay for the diagnosis of TB.</a> N Engl J Med. 356, 188-189 (2007).</li><li>Park, W. G., Bishai, W. R., Chaisson, R. E., Dorman, 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=Performance+of+the+microscopic+observation+drug+susceptibility+assay+in+drug+susceptibility+testing+for+Mycobacterium+tuberculosis.">Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis.</a> J Clin Microbiol. 40, 4750-4750 (2002).</li><li>Shiferaw, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microscopic+observation+drug+susceptibility+assay+for+detection+of+multidrug-resistant+Mycobacterium+tuberculosis.">Evaluation of microscopic observation drug susceptibility assay for detection of multidrug-resistant Mycobacterium tuberculosis.</a> J Clin Microbiol. 45, 1093-1093 (2007).</li><li>Tovar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+diagnosis+of+pleural+tuberculosis+using+the+microscopic-+observation+drug-susceptibility+technique.">Improved diagnosis of pleural tuberculosis using the microscopic- observation drug-susceptibility technique.</a> Clin Infect Dis. 46, 909-909 (2008).</li><li>Vargas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+sputum-scarce+HIV-associated+pulmonary+tuberculosis+in+Lima,+Peru.">Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru.</a> Lancet. 365, 150-150 (2005).</li></ol>

Le test MODS est destiné aux ressources limitées. Pour la première fois, MODS apporte la capacité de détection rapide de la culture liquide de la tuberculose et la tuberculose multirésistante aux ressources limitées à un peu moins de 3 $ par test. MODS est un non-propriétaires, méthodologie itérative, et la communauté MODS est toujours intéressé à l&#39;amélioration d&#39;autres laboratoires ont réussi à faire. Une préoccupation récurrente est la biosécurité de la culture des milieux liquides de la tuberculose,...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Refrigerator/ freezer</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to store pre-prepared broth and antibiotic stocks</td>
</tr>
<tr>
<td>Vortex</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to aid sputum decontamination</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed)</td>
</tr>
<tr>
<td>Incubator (37 degree C)</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for culture; need not be CO2 enriched</td>
</tr>
<tr>
<td>Inverted light microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to read MODS plates</td>
</tr>
<tr>
<td>Autoclave</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to sterilize media, PBS and used plates</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to weigh isoniazid, rifampicin and NALC</td>
</tr>
<tr>
<td>Middlebrook 7H9 broth (Difco)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0713-17-9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Casitone (pancreatic digest casein)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0259&#x2010;17&#x2010;9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Glycerol (glycerin) lyophilized</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G&#x2010;33&#x2010;500</td>
<td>500ml/bottle; culture media base</td>
</tr>
<tr>
<td>PANTA (Antibiotic mixture lyophilized BD) </td>
<td>Reagent</td>
<td>Fisher</td>
<td>B4345114</td>
<td>6 bottles/pack; antibiotic media supplement</td>
</tr>
<tr>
<td>OADC (Middlebrook OADC enrichment BD)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>B11886</td>
<td>10 x 20ml/pack; nutritional media supplement</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (Hibri-Max)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D-2650</td>
<td>100ml/bottle; to prepare rifampicin stock</td>
</tr>
<tr>
<td>Antibiotic stocks: isoniazid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I-3377</td>
<td>50gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Antibiotic stocks: rifampicin</td>
<td>Reagent</td>
<td>Sigma 99</td>
<td>R-3501</td>
<td>1gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Sodium hydroxide (pellets)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>221465</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium citrate (trisodium salt dihydrate)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S-4641</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>N-acetyl-L-cysteine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7250</td>
<td>50gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Potassium Phosphate Monobasic crystal. KH2PO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0662</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium Phosphate Dibasic, anhydrous. Na2HPO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0876</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium hypochlorite</td>
<td>Reagent</td>
<td>household bleach</td>
<td>&nbsp;</td>
<td>to discard contaminated waste</td>
</tr>
<tr>
<td>15ml centrifuge tubes (polypropylene 15ml Falcon 35&#x2010;2096)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>14&#x2010;959&#x2010;49B</td>
<td>500ea/case; for sputum decontamination and concentration</td>
</tr>
<tr>
<td>24 well plates (Plates Tissue 24 wells BD Falcon 35&#x2010;3047)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>50 plates/case; for culture and reading</td>
</tr>
<tr>
<td>Sealable polythene bags 6 X 6 &#x201C; (ziplock)</td>
<td>Consumable</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for biosecurity to contain 24 well plate</td>
</tr>
<tr>
<td>Glass tubes with lid (16 x 100mm and 18 x 145mm)</td>
<td>Consumable</td>
<td>VWR</td>
<td>47729-583</td>
<td>500 tubes/case; to store aliquots of prepared broth</td>
</tr>
<tr>
<td>Screw cap microcentrifuge tubes (1.5ml)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>05&#x2010;669&#x2010;22</td>
<td>1000ea/case; to store aliquots of antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (aqueous solvents) Syringe filter Millex blue</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGL 025 OS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (organic solvents) Syringe filter Millex yellow</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGV 033 RS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>Disposable Pasteur pipettes borosilicate glass 9&#x2033;</td>
<td>Consumable</td>
<td>Fisher</td>
<td>13&#x2010;678&#x2010;20C</td>
<td>720ea/case; to mix PANTA with media mix</td>
</tr>
<tr>
<td>Aerosol barrier tips 1000&#x2010;1300&#x03BC;l</td>
<td>Consumable</td>
<td>Fisher</td>
<td>02&#x2010;707&#x2010;51</td>
<td>1000ea/pk; to dispense media into plate</td>
</tr>
<tr>
<td>USA Scientific Tips One 1&#x2010;200&#x03BC;l yellow tips</td>
<td>Consumable</td>
<td>Fisher</td>
<td>1111&#x2010;0006</td>
<td>1000 tips/bag; to dilute antibiotic stocks</td>
</tr>		</tbody>
		</table>
		</div>

the mods method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Les patients atteints de tuberculose pulmonaire (TB) active infecter d&#39;autres personnes de 10 à 15 par an, ce qui rend le diagnostic de tuberculose active essentielle à la fois à guérir le patient et la prévention des nouvelles infections. Par ailleurs, l&#39;émergence de la tuberculose multirésistante (tuberculose à bacilles multirésistants) signifie que la détection de la résistance aux médicaments est nécessaire pour arrêter la propagation de souches résistantes aux médicaments. L&#39;observation microscopique-sensibilité aux médicaments (MODS) de dosage est un faible coût, faible technicité outil de haute performance de détection de la tuberculose et TB-MR. Le test MODS est basé sur trois principes: 1) de Mycobacterium tuberculosis (MTB) croît plus vite dans les milieux liquides que sur milieu solide 2) la croissance VTT microscopiques peuvent être détectés plus tôt dans les milieux liquides que d&#39;attendre que l&#39;aspect macroscopique des colonies sur milieu solide, et que la croissance est caractéristique de VTT, qui lui permet d&#39;être distinguée de la contamination mycobactéries ou fongiques ou bactériennes atypiques 3) les médicaments isoniazide et la rifampicine peuvent être incorporés dans le test MODS pour permettre une détection directe simultanée de tuberculose à bacilles multirésistants, éliminant la nécessité d&#39;une sous-culture à effectuer un test de drogue indirects susceptibilité. La concurrence des diagnostics actuels sont entravés par une faible sensibilité aux frottis de crachats, de longs délais jusqu&#39;à ce diagnostic avec la culture médiatique solide, avec des coûts prohibitifs des méthodes existantes de liquide milieux de culture, et la nécessité de ne sous-culture pour les tests de sensibilité aux médicaments indirectes pour détecter la tuberculose multirésistante. En revanche, la méthode non-propriétaires MODS a une sensibilité élevée pour la tuberculose et tuberculose à bacilles multirésistants, est une méthode de culture relativement rapide, offre simultanée des tests de sensibilité aux médicaments pour la tuberculose multirésistante, et est accessible aux ressources limitées à un peu moins 3 $ pour les tests pour la tuberculose et TB-MR.

Watch this Scientific Journal Video about The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis at JoVE.com

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

L&#39;observation microscopique-sensibilité aux médicaments (MODS) de dosage est un faible coût, faible technicité outil de haute performance de détection de la tuberculose (TB) et la tuberculose multirésistante (tuberculose à bacilles multirésistants). Cette vidéo décrit le liquide MODS la méthode de culture des médias.

La méthode MODS pour le diagnostic de la tuberculose et la tuberculose multirésistante

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

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18.9K vues.  The Warren Alpert Medical School of Brown University. L'observation microscopique-sensibilité aux médicaments (MODS) de dosage est un faible coût, faible technicité outil de haute performance de détection de la tuberculose (TB) et la tuberculose multirésistante (tuberculose à bacilles multirésistants). Cette vidéo décrit le liquide MODS la méthode de culture des médias.

Vidéo: The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Injection d&#39;un. Embryons stephensi pour générer le paludisme résistant à moustiques

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

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Biologie

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expression, Solubilisation détergent, et purification d&#39;un transporteur membranaire, la protéine MexB multirésistance

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

La méthode de culture des embryons rongeurs entier en utilisant le système des rotateurs de type flacon de culture

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

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

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

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

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Une méthode de génotypage basé sur la PCR pour distinguer les variétés de type sauvage et les plantes ornementales de 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 ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Haut débit cristallisation des protéines membranaires en utilisant la méthode lipidique Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Une méthode simple pour l&#39;imagerie Arabidopsis Feuilles utilisant perfluorodécaline comme un moyen d&#39;imagerie infiltratives

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Le / GFP-FLP Méthode tomate / FRT pour l&#39;imagerie en direct de mosaïque adulte<em&gt; Drosophila</em&gt; cellules photoréceptrices

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Un procédé de criblage et de validation des mutations de résistance aux inhibiteurs de kinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Méthode d&#39;évaluation des effets d&#39;une gamme de longueurs d&#39;onde et des intensités de Luminothérapie Rouge / proche infrarouge sur le stress oxydatif<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Filtration Isolation of Nucleic Acids:  A Simple and 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 to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolement des acides nucléiques: Une méthode d&#39;extraction de l&#39;ADN simple et rapide

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