Biz Sean Fitzwater ve Carmen Giannina Luna Colombo tüberküloz büyüme time-lapse video segment için kabul etmek istiyorum. Biz eksiksiz ve mükemmel bir geri bildirim için geçerli protokol çoğunlukla birebir alındığı Kullanım Kılavuzu, düzenleme sırasında Marty Roper teşekkür ediyorum ve co-yazma. Bu video Üretim NIH / Fogarty Uluslararası Merkezi tarafından finanse edildi <a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a> David AJ Moore Imperial College London Bulaşıcı Hastalıklar Tropikal Tıp ve Reader Wellcome Trust Klinik Araştırma Görevlisi (Fellowship olarak katkıda ödül sayısı 078067/Z/05). Mark F. Brady bir NIH / Fogarty Uluslararası Araştırma Merkezi Araştırma Görevlisi olarak katkıda bulundu.

<ol><li> Stok çözelti hazırlayın <ol><li> Fosfat tampon stok <ol><li> , Potasyum fosfat tekbazlı çözüm 950ml (1000ml distile su içinde çözünmüş potasyum fosfat tekbazlı 9.07g) ve karıştırın, sodyum dibazik çözüm 950ml (1000ml distile su içinde çözünmüş sodyum fosfat dibazik 9.47g) karıştırın; ayarlamak için her çözümün geri 50ml tutabilecek pH gerekli özelliğine </li><li> PH 6.8 ± 0.2 ayarlayın: pH yükseltmek eklemek sodyum fosfat dibazik çözümü; düşük pH potasyum fosfat tekbazlı çözüm eklemek </li><li> 121-124 az Otoklav ° C&#39;de 15 dakika sterilize </li><li> Plaka 48 saat için 37 Petri kabı ve inkübe besin agar fosfat tampon çözeltisi ° C 100μl kısım kısırlık onaylamak için </li><li> Buzdolabında saklayın 2-8 ° C için bir ay kadar Not: Her balgam örneği fosfat tampon çözelti 10ml gerektirir . </li></ol></li><li> Dekontaminasyon stok <ol><li> Kombine sodyum hidroksit çözeltisi eşit hacimlerde (200 ml steril distile su içinde çözünmüş sodyum hidroksit 8.0g) ve sodyum sitrat solüsyonu (5.8g, 200 ml steril distile su distile sodyum sitrat) </li><li> 121-124 ° C&#39;de 15 dakika boyunca karıştırın ve otoklav </li><li> Buzdolabında saklayın 2-8 ° C için bir ay kadar Not: Her bir balgam örneği dekontaminasyon stokunun 2ml gerektirir </li></ol></li><li> Kültür medya stok <ol><li> Steril distile su 900ml içine gliserol ve casitone 7H9 orta toz ve 5.9g 1.25g 3.1ml ekleyin; tamamen eriyene kadar sürekli ajitasyon ile karıştırın </li><li> 15 dakika süreyle 121-124 ° C&#39;de Otoklav </li><li> Serin ve antibiyotik çözümleri ve iç kontrollerin hazırlanması için steril numune hazırlama için 16x 100 mm cam tüpler ve 10.8ml alikotları 4.5ml alikotları içine steril ortamda bölmek </li><li> Kısırlık (bulanıklık eksikliği) doğrulamak için 37 ° C&#39;de 48 saat süreyle inkübe </li><li> 2-8 ° C kap ile saklayın sıkı bir ay kadar kapalı Not: Her bir balgam örneği 7H9 orta bir tüp içeren 4.5ml gerektirir. </li></ol></li><li> Antibiyotik stok çözümleri <ol><li> A. İzoniyazid stok (8 mg / ml) <ol><li> 20 mg 2.5ml steril distile su tamamen izoniazid çözülür </li><li> Sulu solvent 0.2μm şırınga filtresi ile filtreleme </li><li> 6 aya kadar -20 ° C&#39;de steril mikro santrifüj tüpleri 20μl alikotları saklayın Not: Her saklanan 20μl kısım 100 numune (fire dahil) için yeterli </li></ol></li><li> B. Rifampisin stok (8 mg / ml) </li></ol><ol><li> 20 mg rifampisin 1.25ml DMSO tamamen çözülür </li><li> 1.25ml steril distile su eklenir ve karıştırılır </li><li> Organik çözücü 0.2μm şırınga filtresi ile filtreleme </li><li> 6 aya kadar -20 ° C&#39;de steril mikrosantrifüj tüpler 20μl alikotları saklayın Not: Her saklanan 20μl kısım 100 numune (fire dahil) için yeterli </li></ol></li></ol></li><li> Çalışma çözümler hazırlayın <ol><li> Dekontaminasyon çalışma çözümü <ol><li> NALC kristalleri gerekli dekontaminasyon çözüm her 20ml 0.1g çözülür Not: Her bir örnek dekontaminasyon çözüm 2ml ihtiyacı Not: NALC mukolitik faaliyet zaman içinde kaybeder gibi 24 saat sonra kullanılmayan kalan NaOH-NALC dekontaminasyon çözümü atın </li></ol></li><li> Kültür medya çalışma çözüm <ol><li> Şu şekilde anlatılır: <ol><li> 7H9 orta her balgam örneği için 1 tüp işlenmiş olması, artı her plaka için 1 tüp (negatif kontrol sütun için) </li><li> 7H9 orta pozitif kontrol için 2 tüp (3 monoresistant suşları kullanılmıştır) </li><li> 10.8ml 7H9 antibiyotik çözeltisi ile 1-2 tüpler </li></ol></li><li> Her bir örnek tüp 7H9 4.5ml 0.5 ml OADC </li><li> 10.8ml 7H9 tüpler 1.2ml OADC </li><li> (Bu PANTA gerekmez) antibiyotik çözelti hazırlama için kullanılacak 12ml 7H9 OADC (ler) pozitif kontrol için 5ml 7H9-OADC ve tüp 2 tüpleri kenara koyun </li><li> PANTA sulandırın ve her numune tüpü ve negatif kontrol tüpleri (7H9-OADC-PANTA: toplam hacim = 5.1ml) 0.1ml eklemek Not: PANTA (7H9-OADC-PANTA) ile tam orta balgam örnekleri ve negatif kontroller için kullanılır; pozitif kontrol ve antibiyotik çözeltisi PANTA olmadan 7H9-OADC </li></ol></li><li> Antibiyotik çalışma çözümleri <ol><li> 1. Kullanılmayan bir 24 plaka antibiyotik çalışma çözümü için adımlar için Bkz: Şekil 1 Not: doğru bir duyarlılık sonuç için, son antibiyotik konsantrasyonları kritik öneme sahiptir. Aşağıdaki yordam l için uygundur.aboratories mikropipetler ile donatılmıştır. Mikropipetler mevcut değilse, tüberkülin şırınga Kılavuzu Ek 3 modsperu.org Kullanıcı açıklanan dilüsyonları farklı bir dizi ile kullanılabilir Not: yeniden dondurmak veya ilaç aktivitesi yok olabilir antibiyotik çalışma veya hisse senedi çözümleri yeniden kullanımı, işlem günü Şekil 1 sonunda tüm kullanılmayan antibiyotik çözümler atın . Antibiyotik çalışma çözümler (modsperu.org Kullanım Kılavuzu) </li></ol></li></ol></li><li> Balgam örneği ve plaka hazırlama <ol><li> Arındırma <ol><li> 15ml bir santrifüj tüpü içine balgam örneği Yeri 2ml (eğer daha az, 2 ml fosfat tamponu ile makyaj daha varsa, sadece 2ml kullanın) </li><li> 2ml NaOH-NALC çözüm ekle </li><li> Kap sıkıca tüp ve 20 saniye boyunca girdap; NaOH-NALC çözüm kişiler, tüm iç tüp yüzey ve kapak sağlamak için tüp ters </li><li> En az 15 dakika bekletin örnek özellikle viskoz ise bir kaç dakika uzatabilir. Tedavi üzerinde önlemek için, 20 dakikayı geçmemelidir </li><li> 4 kez alkali nötralize ve dekontaminasyon işlemi sona erdirmek ve tüpü ters çevirerek iyice karıştırın fosfat tamponu (pH 6.8) ile 14ml tüp doldurun </li><li> 3000 g de 15 dakika süreyle santrifüj (3000 g ulaşmak için gerekli dakikadaki dönüş denklemi için modsperu.org Kullanım Kılavuzu Ek 2) </li><li> Dikkatlice bir sıvı atık kap içine% 10 sodyum hipoklorit veya diğer uygun bir dezenfektan ile süpernatant kapalı dökün ve pelet korumak </li></ol></li><li> Son örneği süspansiyon ve yedekleme hazırlanması <ol><li> 7H9-OADC-PANTA (5.1ml içeren tüp), Pasteur pipeti ile santrifüj tüpüne toplam hacmi 2 ml örnek pelet tekrar süspansiyon, iyice karıştırın </li><li> Yedek olarak 2-8 örnek bir mikrosantrifüj tüp içinde süspansiyon ve mağaza ° C 1 ml çıkarın </li><li> Ikinci 1ml örnek süspansiyon kalan 7H9-OADC-PANTA ile tüp ekleyin, iyice karıştırın. Bu kaplama için hazır son örnek çözümdür </li></ol></li><li> 24-plaka örnek süspansiyonlar yerleştirilmesi <ol><li> 24 plaka bir sütun 4 kuyuların her birine son örnek süspansiyon Yeri 900μl </li><li> Sütun 3 ek örnekler dışında, plaka tüm sütunlar kadar tekrarlayın (ya da tüm örnekler kaplama kadar) doldurulur </li><li> Her numune plaka Sütun 3 4 kuyu (negatif iç kontrollerin) örnek olmadan 7H9 OADC orta Yeri 900μl </li></ol></li><li> Örnekler antibiyotik ekleme <ol><li> Çok kanallı pipet kullanarak, dikkatli bir şekilde 7H9-OADC ve antibiyotik çalışma çözümleri (Sütun 2 antibiyotik seyreltme plaka Şekil 1) ile kuyulardan 100μl doldurmak 4 ipucu </li><li> Levha veya örnek dokunmadan 900μl örnek çözümleri ile sütun 1 kuyu 100μl alikotları ekle </li><li> Tüm sütunları orta ek 100μl (ilaçsız kuyular) veya (negatif kontrol sütun 3 de dahil olmak üzere) antibiyotik çözümler aldık kadar bu işlemi tekrarlayın </li><li> Yapışmalı, polietilen (KİLİTLİ) çanta ve mühür kapak ve yer ile plaka kapatın (çanta Bu noktadan itibaren tekrar açıldı) </li><li> 37 ° C (CO 2 zenginleştirme gerekli değildir) inkübe </li></ol></li></ol></li><li> Kalite kontrolü <ol><li> Negatif kontroller, her bir plaka üzerinde çalıştırılır. Negatif kontrol sütununda herhangi bir kuyu herhangi bir büyüme varlığı, çapraz kontaminasyon, bu nedenle tüm plaka atılmalıdır ve örnekleri yeniden kaplama gösterir. </li><li> Pozitif kontrolleri günün sonunda ayrı bir plaka üzerinde çalıştırılır. Bir tümüyle ilaçlara duyarlı gerginlik, bir sütun ve başka bir sütun çalıştırmak INH ve RIF bir yük dayanıklı çalıştırılır . MDR gerginlik çalıştırma hakkında endişe, INH monoresistant gerginlik hem RIF monoresistant suşu duyarlı ve MDR suşlarının yerde çalıştırılabilir varsa. Tüm pozitif kontrol medya desteği büyüme göstermek için uyuşturucu ücretsiz kuyularda büyüme olmalıdır. Ilaç konsantrasyonları doğru olduğunu göstermek için, duyarlı bir gerginlik INH veya RIF kuyuları büyümek değil, ancak MDR gerginlik INH ve RIF kuyuları da büyümeye büyümek gerekir. </li></ol></li><li> Plaka okuma <ol><li> &quot;Gün 0&quot; olarak kaplama gün ile plaka okuma 5. gününde başlar, ve eğer negatifse Günü&#39;nde okuma 5 gün 14 ve 15 gün sonra her 2-3 gün kadar (ya da iş yüküne bağlı olarak alternatif gün) her gün yapılıyor -21. Tabaklar bir incelenir10x hedefi ile ters ışık mikroskobu </li><li> Olumlu bir kültür İKİ uyuşturucu ücretsiz kuyulardan iki veya daha fazla koloni oluşturan birim (2 CFU) var olan biridir. Erken mikobakteriyel büyüme küçük kavisli virgül veya spiral (5-9 gün) gibi görünüyor. Colony oluşumu genellikle &quot;kordonlar&quot; veya &quot;Tangles&quot; (modsperu.org görüntü kütüphanesi) ilerler </li><li> Günü uyuşturucu içeren kuyu okunacağı 2 kuyu, uyuşturucu ücretsiz bir kültür pozitif bir ilacın varlığında herhangi bir büyüme bu ilaca karşı direnç gösterir . Not: Ara sıra uyuşturucu ücretsiz kuyularda pozitif bir kültür olduğunu kaydetti&gt; 1 hafta sonra ilaç içeren kuyuların okuma yapılırsa, bu büyüme break-direnci temsil olmayabilir görülür </li><li> Gün 21 ile pozitif numune negatif kültürler kabul edilir </li><li> Mikobakteriyel büyüme bulutluluk sonuç vermez ise kuyuların bulutluluk, kirlenme aşırı çoğalma sonucu </li><li> 21 gün tamamlanması kültürler atılabilir. Kültürler mühürlü torbalar içinde kalır ve otoklav torbaları yerleştirilir ve 45-60 dakika süreyle 121-124 ° C&#39;de otoklava </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>

MODS tahlil, kaynak sınırlı ayarları hedeflenmektedir. Ilk kez, MODS tüberküloz hızlı sıvı kültür algılama yeteneği getiriyor ve hemen altında test başına 3 $ kaynak sınırlı ayarları dirençli tüberküloz ilaca. MODS ticari olmayan, iteratif metodoloji ve MODS topluluk her zaman diğer laboratuvarlardan yapmak başarmış iyileştirmelerini de ilgileniyor. Sıvı dökülürse veya aerosolize olabilir, çünkü tüberküloz sıvı medya kültürü, nükseden bir endişe biyogüvenlik. MODS testi dolaylı olarak ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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

Aktif akciğer tüberküloz (TB) olan hastalar, hasta kür ve yeni enfeksiyonların önlenmesi için gerekli aktif verem teşhis yapma, yılda 10-15 kişi enfekte. Ayrıca, çok ilaca dirençli tüberküloz (MDRTB) ortaya çıkması, ilaç direncinin saptanması ilaca dirençli suşların yayılmasını durdurmak için gerekli anlamına gelir. Mikroskopik gözlem ilaç duyarlılık (MODS) testi, TB ve MDRTB yüksek performanslı bir algılama için düşük maliyetli, düşük teknolojili bir araçtır. Mycobacterium tuberculosis (MTB) mikroskobik MTB büyüme katı ortam kolonilerinin makroskopik görünüm için beklemek yerine sıvı ortamda daha önce tespit ve olabilir) katı ortam 2 daha sıvı ortamda hızlı büyür 1): MODS testi üç temel ilkeye dayanır büyüme altkültürü gerçekleştirmek için ihtiyaç kalmayabilir, MTB karakteristik ilaçlar izoniazid ve rifampisin MDRTB eş zamanlı olarak doğrudan tespiti için izin MODS testi dahil edilebilir atipik mikobakteriler veya mantar veya bakteriyel kontaminasyon 3) ayırt edilmesi için izin dolaylı bir ilaç duyarlılık testi. Rekabet mevcut teşhis balgam smear ile düşük hassasiyet, katı ortam kültürü, mevcut sıvı ortam kültür yöntemleri ile yasaklı yüksek maliyet ve MDRTB tespit etmek için dolaylı olarak ilaç duyarlılık testi için altkültürü yapmak ihtiyacı ile tanıya kadar uzun gecikmeler engel. Buna karşılık, ticari olmayan MODS yöntemi, TB ve MDRTB için bir yüksek hassasiyet vardır bir görece hızlı kültür yöntemi, MDRTB için eş zamanlı olarak ilaç duyarlılık testi sağlar, ve TB için test için sadece altında ABD doları 3 kaynak sınırlı ayarları erişilebilir ve MDRTB.

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

Mikroskopik gözlem ilaç duyarlılık (MODS) tayini, düşük maliyetli, yüksek performanslı bir algılama tüberküloz (TB) ve çoklu ilaca dirençli tüberküloz (MDRTB) düşük teknolojili bir araçtır. Bu video MODS sıvı medya kültürü yöntemi açıklanmıştır.

MODS tüberküloz tanısı için yöntem ve dirençli tüberküloz ilaca

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

the-mods-method-for-diagnosis-tuberculosis-multidrug-resistant

Research

JoVE Journal

Biology

18.9K Görüntüleme.  The Warren Alpert Medical School of Brown University. Mikroskopik gözlem ilaç duyarlılık (MODS) tayini, düşük maliyetli, yüksek performanslı bir algılama tüberküloz (TB) ve çoklu ilaca dirençli tüberküloz (MDRTB) düşük teknolojili bir araçtır. Bu video MODS sıvı medya kültürü yöntemi açıklanmıştır.

Video: 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.

Bir, Enjeksiyon. stephensi Embriyolar Sıtma dayanıklı Sivrisinekler oluşturun

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

Biyoloji

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.

İfade, Deterjan çözünürleştirme ve Membran Transporter, MexB ilaca Direnç Protein Saflaştırılması

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.

Rotator-tipi Şişe Kültür Sistemi kullanılarak Kemirgen Tüm Embriyo Kültürü Yöntemi

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).

Wild-tip ve Süs Çeşitler arasında ayırt A PCR tabanlı Genotiplendirme Yöntemi 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.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

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.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

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.

Mozaik Yetişkin Canlı Görüntüleme Domates / GFP-FLP / FRT Yöntemi<em&gt; Drosophila</em&gt; Fotoreseptör Hücreler

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.

Kinaz İnhibitörleri Karşı Dayanıklı Mutasyonlarının Tarama ve Doğrulama için Bir Yöntem

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.

Dalga boyundaki ve Oksidatif Stres Kırmızı / yakın-kızılötesi ışık Terapisi şiddetler bir Range Etkilerinin Değerlendirilmesi için Yöntemi<em&gt; İn 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.

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

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