우리는 결핵의 성장 시간 저속 비디오 세그먼트에 대한 숀 피츠 워터와 카르멘 Giannina 루나 콜롬보을 인정하고 싶습니다. 우리는 현재의 프로토콜은 대부분 그대로 찍은있는 사용자 가이드를 편집하는 동안 그녀의 철저하고 탁월한 의견에 마티 로퍼 감사 및 공동 제작. 이 비디오의 제작은 NIH / 포가티 국제 센터에 의해 투자되었다 <a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a> 데이비드 AJ 무어 임페리얼 칼리지 런던 대학에서 전염병에 열대 의학 및 리더의 웰컴 트러스트 임상 연구원 (펠로우쉽으로 기여 보너스 번호 078067/Z/05). 마크 F. 브래디는 NIH / 포가티 국제 센터 연구원으로 기여.

<ol><li> 재고 솔루션을 준비 <ol><li> 인산 버퍼 주식 <ol><li> 인산 칼륨 일염기의 솔루션 950ml (1000ml 증류수에 녹아있는 인산 칼륨 일염기의의 9.07g)과 볶음과 함께 나트륨 이염 솔루션의 9​​50ml를 (1000ml 증류수에 녹아있는 인산 나트륨의 이염의 9.47g) 혼합, 조정 각 솔루션의 뒤로 50ml 유지 산도 필요한 경우 </li><li> 6.8 ± 0.2 산도를 조정 : 산도 마련에 추가 인산 나트륨 이염 솔루션, 낮은 산도로 인산 칼륨 일염기의 솔루션을 추가 </li><li> 121-124에서 압력솥 ° C 15 분 소독하기 </li><li> 플레이트 48 시간 37 페트리 접시와 부화에 영양소 한천 배지에 인산 버퍼 용액 ° C의 100μl 나누어지는가 불임을 확인하기 </li><li> 2-8에서 냉장고에 보관 ° C에 1 개월 참고 : 각 가래 샘플은 인산 버퍼 용액 10ml을 필요로 </li></ol></li><li> 오염 제거 주식 <ol><li> 동일한 수산화 나트륨 솔루션의 볼륨 (200ml 멸균 증류수에 녹아 수산화 나트륨의 8.0g)과 나트륨 구연 산염 솔루션 (200ml 멸균 증류수에 소주 나트륨 구연 산염의 5.8g) 결합 </li><li> 15 분 동안 121-124 ° C에서 믹스 앤 압력솥 </li><li> 2-8에서 냉장고에 보관 ° C에 1 개월 참고 : 각 가래 샘플은 방사능 제거 주식 ​​2ml 필요 </li></ol></li><li> 문화 미디어 주식 <ol><li> 멸균 증류수의 900ml에 글리세린과 7H9 중간 가루 casitone과 5.9g의 1.25g의 3.1ml 추가, 지속적인 교반과 혼합 완전히 해산까지 </li><li> 15 분 동안 121-124 ° C에서 압력솥 </li><li> 쿨 및 항생 솔루션과 내부 통제의 준비를위한 무균 샘플 준비 16X 100mm 유리 튜브 및 10.8ml aliquots에 4.5ml aliquots에 멸균 매체를 나누어 </li><li> ° C 48 시간 동안 불임을 (탁​​도의 부족) 확인하기 위해 37 알을 품다 </li><li> 2-8 ° 모자와 C의 저장소가 긴밀하게 1 개월에 폐쇄 참고 : 각 가래 샘플 7H9 매체 중 하나가 포함된 튜브 4.5ml 필요 </li></ol></li><li> 항생제 재고 솔루션 <ol><li> A. Isoniazid 주식 (8 MG / ML) <ol><li> 20 MG는 2.5ml 멸균 증류수에 완전히 용해 isoniazid </li><li> 수성 용매에 대한 0.2μm 주사기 필터와 필터 </li><li> 최대 6 개월까지 -20 ° C에서 살균 마이크로 원심 분리 튜브에 20μl aliquots에 저장 참고 : 각 저장 20μl 나누어지는가 최대 100 개의 샘플 (소모 포함)에 대한 충분 </li></ol></li><li> B. Rifampicin 주식 (8 MG / ML) </li></ol><ol><li> 1.25ml DMSO 완전히 20mg rifampicin을 풀다 </li><li> 1.25ml 무균 증류수와 혼합 추가 </li><li> 유기 용매에 대한 0.2μm 주사기 필터와 필터 </li><li> 최대 6 개월까지 -20 ° C에서 살균 microcentrifuge 튜브에 20μl aliquots에 저장 참고 : 각 저장 20μl 나누어지는가 최대 100 개의 샘플 (소모 포함)에 대한 충분 </li></ol></li></ol></li><li> 작업 솔루션을 준비 <ol><li> 오염 제거 작업 솔루션 <ol><li> 필요한 오염 제거 솔루션의 모든 20ml에 NALC 결정을 0.1g을 풀다 참고 : 각 예제는 오염 제거 솔루션 2ml 요구 참고 : NALC은 시간이 경과함에 따라 mucolytic 활동을 잃는 등 24 시간 후에 사용하지 않는 남아있는 NaOH - NALC의 오염 제거 솔루션을 무시 </li></ol></li><li> 문화 미디어 작업 솔루션 <ol><li> 아웃 설정 : <ol><li> 모든 가래 샘플 7H9 중간 1 튜브 처리, 플러스마다 접시 하나 추가 튜브 (부정적인 제어 열)으로 </li><li> 긍정적인 컨트롤 7H9 중간 2 튜브 (3 monoresistant 종자를 사용하는 경우) </li><li> 항생제 솔루션 준비 10.8ml 7H9과 1-2 튜브 </li></ol></li><li> 각 샘플 튜브 7H9의 4.5ml을 0.5ml OADC 추가 </li><li> 10.8ml 7H9와 튜브에 1.2ml OADC 추가 </li><li> (이들은 PANTA 필요하지 않습니다) 항생제 솔루션 준비에 사용되는 12ml 7H9 - OADC로 (S) 5ml 긍정적인 컨트롤 7H9 - OADC하고, 튜브 2 튜브를 따로 설정 </li><li> Reconstitute의 PANTA 각 샘플 튜브와 부정적인 제어 튜브 (7H9 - OADC - PANTA : 전체 볼륨 = 5.1ml)을 0.1ml 추가 참고 : PANTA (7H9 - OADC - PANTA)와 완전한 매체가 가래 샘플 및 부정적인 제어에 사용되며 긍정적인 컨트롤과 항생 솔루션 준비 PANTA없이 7H9 - OADC 사용 </li></ol></li><li> 항생제 작업 솔루션 <ol><li> 1. 사용하지 않는 24 - 잘 판에 항생제 작업 솔루션을 제작하는 단계에 대한 그림 1을 참조 참고 : 정확한 자화율 결과, 최종 항생제 농도가 중요합니다. 다음 절차는 리터에 적합합니다aboratories는 micropipettes 갖춘. micropipettes를 사용할 수없는 경우, 투베르쿨린 주사기는 modsperu.org에서 가이드 부록 3 사용자에 설명되어 dilutions의 다른 시리즈와 함께 사용할 수 있습니다 참고 :하지 마십시오 다시 동결 또는 마약 관련 활동은 유실될 수 있으므로 항생제 작업 또는 주식 솔루션을 다시 사용하는 처리 하루 그림 1의 끝 부분에 모두 사용하지 않는 항생제 솔루션을 폐기하십시오.. (modsperu.org에서 사용자 설명서에서) 항생제 작업 솔루션을 만들기 </li></ol></li></ol></li><li> 가래 샘플 및 플레이트 준비 <ol><li> 오염을 제거하는 것 <ol><li> 15ml 원심 튜브에 가래 샘플의 장소 2ml (면 이하, 인산 버퍼 2ml까지하게, 더 많은 경우에만 2ml를 사용하십시오) </li><li> 2ml NaOH - NALC 솔루션 추가 </li><li> 캡 밀접하게 관 20 초 소용돌이, NaOH - NALC 솔루션 연락처에게 전체 내부 튜브의 표면과 뚜껑을 보장하기 위해 튜브를 반전 </li><li> 15 분 최소 스탠드하자 - 샘플 특히 점성 경우 몇 분 연장하실 수 있습니다. 치료를 통해 방지를 위해, 20 분 넘지 않아야 </li><li> 4 번 알칼리 중화 및 오염 제거 프로세스를 종료하고, 반전 튜브로 잘 섞어 인산 버퍼 (산도 6.8)와 14ml로 튜브를 채우 </li><li> 15 분 동안 3,000그램에서 원심 분리기 (3천g에 도달하기 위해 필요 분당 회전의 방정식에 대한 modsperu.org의 사용 설명서의 부록 2 참조) </li><li> 조심스럽게 10 % 나트륨 차아 염소 산염 또는 기타 적합한 살균제와 액체 폐기물 컨테이너에 뜨는를 부어 및 펠릿을 유지 </li></ol></li><li> 최종 샘플 정지 및 백업의 준비 <ol><li> 7H9 - OADC - PANTA을 (5.1ml이 들어있는 튜브에서) 사용하면 파스퇴르 피펫으로 원심 튜브에 2ml의 총 볼륨의 샘플 펠렛을 resuspend; 잘 혼합 </li><li> 백업으로 2-8에서 샘플 서스펜션과 microcentrifuge 관에 저장 ° C의 1ml를 제거 </li><li> 나머지 7H9 - OADC - PANTA와 튜브 샘플 중지의 두 번째 1ml를 추가, 잘 섞는다. 이것은 도금을위한 준비 최종 샘플 솔루션입니다 </li></ol></li><li> 24 잘 플레이트로 샘플 suspensions 배치 <ol><li> 24 잘 플레이트의 칼럼의 4 우물의 각에 최종 샘플 정지 플레이스 900μl </li><li> , 열 세를 제외하고 접시의 모든 열 때까지 추가 샘플과 함께 반복 (또는 모든 샘플이 도금까지) 채워집니다 </li><li> 각 시료 접시의 열 3 4 우물 (부정적인 내부 통제)에 샘플 않고 7H9 - OADC 매체 플레이스 900μl </li></ol></li><li> 샘플로 항생제 추가 <ol><li> 멀티 채널 피펫을 사용하여 조심스럽게 7H9 - OADC 및 항생 작업 솔루션 (항생제 희석의 열 두 그림 1 판 - 참조)와 우물에서 100μl 4 개의 도움말을 기입 </li><li> 판이나 샘플을 건드리지 않고도 900μl 샘플 솔루션을 열 한 우물에 100μl aliquots 추가 </li><li> 모든 열이 매체의 추가 100μl (약물이없는 우물) 또는 (대조군 열 세 포함) 항생제 솔루션을받은 때까지 반복 </li><li> sealable polythene (애) 가방 및 인감 년에 덮개 및 장소 접시를 닫습니다 (가방이 포인트 이후 다시 오픈되지 않습니다) </li><li> 37 ° C (CO 2 농축이 필요하지 않습니다)에 품어 </li></ol></li></ol></li><li> 품질 관리 <ol><li> 부정적인 통제는 각 접시에 실행됩니다. 대조군 열에있는 우물의 어떤 성장의 존재가 교차 오염, 따라서 전체 접시가 폐기되어야하고 샘플을 다시 도금을 나타냅니다. </li><li> 양성 컨트롤이 하루의 끝에 별도의 접시에 실행됩니다. 하나는 완벽하게 약물 민감한 변형이 열이 다른 열에 실행 INH와 RIF에 스트레인 방지에서 실행됩니다. MDR 스트레인 실행에 대한 우려, INH monoresistant 변형 모두와 RIF monoresistant 변종이 감염될와 MDR 긴장의 장소에서 실행할 수있다면. 모든 긍정적인 컨트롤하는 미디어 지원 성장을 증명하기 위해 약물없는 우물 성장이 있어야합니다. 약물 농도가 올바른지 입증하려면 민감한 긴장은 INH RIF이나 우물에서 성장하지 말아야하지만, MDR 스트레인은 ​​INH와 RIF 우물도 성장 성장해야합니다. </li></ol></li><li> 플레이트 읽기 <ol><li> 로 도금의 하루 &quot;일 0,&quot;판 읽기 5 일에 시작하고, 부정적인 경우에는 주 5 독서는 일 14 다음 일 15 매 2~3일 때까지 (또는 작업 부하에 따라 대체 일간) 매일 이루어집니다 -21. 접시가에 대한 검사 아르10X 목적으로 바뀌 가벼운 현미경 </li><li> 긍정적인 문화는 마약없는 우물 둘 다에서 두 개 이상의 콜로니 형성 단위 (&gt; 2 CFU)가있는 하나입니다. 조기 mycobacterial 성장은 작은 곡선이나 컴마 나선 은하 (일 5-9) 같다. 콜로니 형성은 일반적으로 &quot;코드&quot;또는 &quot;꼬인 것&quot;(modsperu.org에 이미지 라이브러리를 참조) 진행 </li><li> 당일 문화 약물 함유 우물이 읽어야 2 약물없는 우물에 긍정적인 것입니다. 약물의 존재의 모든 성장은 약물에 대한 저항력을 나타냅니다. 참고 : 긍정적인 문화가 마약없는 우물에 기록됩니다&gt; 일주 후 약물이 포함된 웰스의 읽는이 완료되면,이 성장 브레이크를 통해 같은 저항을 대표하지 않을 수도 가끔 볼 수 있습니다 </li><li> 일 21에 의해 긍정하지 않습니다 샘플은 부정적인 문화로 간주됩니다 </li><li> mycobacterial 성장 흐려 발생하지 않는 반면, 우물의 흐려은 오염 전면에 자라난의 결과입니다 </li><li> 21일 완료에 문화가 삭제할 수 있습니다. 문화는 밀폐 봉지에 남아 압력솥 가방에 위치하고 45-60분에 대한 121-124 ° C에서 autoclaved 아르 </li></ol></li></ol>

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	<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>

MODS의 분석은 자원 제한 설정 대상으로합니다. 처음으로, MODS은 결핵의 빠른 액체 문화 감지 기능을 제공하고 테스트 당 단지 달러 이하 3 자원 제한 설정에 대한 내성 결핵을 multidrug. MODS이 아닌 독점적인 반복 방법론이며, MODS 커뮤니티는 항상 다른 실험실 만들 수 있었다 것을 개선에 관심을 가지고있다. 액체가 쏟았거나 aerosolized 수 있기 때문에 회귀 우려가 결핵의 액체 미디어 문화의 biosafety입니다. 우?...

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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

활성 폐 결핵 환자 (TB)는 환자를 치료하고 새로운 감염을 예방 모두에 필수적인 활성 TB를 진단하고, 매년 10-15 다른 사람을 감염. 또한, 강한 결핵 (MDRTB)를 multidrug의 출현은 약제 내성의 검색이 약물 모성 변종의 확산을 막을 필요가있다는 것을 의미합니다. 현미경 - 관찰 약물 자화율 (MODS) 분석 결핵 및 MDRTB의 고성능 감지를위한 저비용, 낮은 기술 도구입니다. mycobacterium 결핵 (MTB)는 미세한 MTB의 성장은 고체 미디어 콜로니의 매크로 모양을 기다리고 이전 액체 미디어 감지하고, 수) 고체 미디어 2 이상의 액체 매체에서 빠르게 성장 1) : MODS 분석은 세 원칙을 기반으로합니다 그 성장은 subculture가 수행의 필요성을 obviating, 그것이 마약 isoniazid와 rifampicin이 MDRTB 동시에 직접 감지 수 있도록 MODS 분석에 통합할 수있는 비정형 mycobacteria이나 곰팡이 또는 세균 오염 3)에서 구분할 수 있도록, MTB의 특징입니다 간접 약물 자화율 시험. 경쟁 현재 진단은 가래 세포진 검사와 함께 낮은 감도, 고체 미디어 문화, 기존의 액체 미디어 문화 방식과 prohibitively 높은 비용, MDRTB를 탐지하기 위해 간접 약물 자화율 테스트 subculture을 필요로 진단을 때까지 긴 지연에 의해 방해하고 있습니다. 반면, 비 - 독점 MODS 방법은, 결핵 및 MDRTB에 대한 높은 감도를 가진 상대적으로 급속한 문화 방식입니다 MDRTB 동시 약물 자화율 시험을 제공하고, 결핵에 대한 테스트를 위해 단지 달러 이하 3 자원 제한 설정에 액세스할 수 있으며 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

현미경 - 관찰 약물 자화율 (MODS) 분석은 결핵의 고성능 감지 (TB)과 multidrug 모성 결핵 (MDRTB)에 대한 낮은 비용, 낮은 기술 도구입니다. 이 동영상은 MODS 액체 미디어 문화 방법을 설명합니다.

MODS의 결핵의 진단 방법과 내성 결핵을 multidrug

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 조회수.  The Warren Alpert Medical School of Brown University. 현미경 - 관찰 약물 자화율 (MODS) 분석은 결핵의 고성능 감지 (TB)과 multidrug 모성 결핵 (MDRTB)에 대한 낮은 비용, 낮은 기술 도구입니다. 이 동영상은 MODS 액체 미디어 문화 방법을 설명합니다.

비디오: 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.

의 사출. stephensi의 태아는 말라리아 방지 모기를 생성하는

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

연구

생물학

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.

표현, 세제 Solubilization 및 멤브레인 전송, MexB의 Multidrug 저항 단백질의 정제

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.

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

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).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 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.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

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.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

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.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

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.

키나제 억제제에 대해 내성 돌연변이의 검사 및 검증을위한 방법

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.

파장과 산화 스트레스에 레드 / 근적외선 치료의 강렬의 범위의 효과의 평가를위한 방법<em&gt; 체외</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.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

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