이 프로토콜의 일부 세부 사항은도 원고에 도움이되는 의견을 제공 박사 아담 돕슨의 도움으로 최적화되었다. 이 작품은 국립 보건원에 대한 재단에 의해 지원되었다 (FNIH) 허가 번호 R01GM095372 (JMC, A (CN) W, AJD 및 AED). FNIH 허가 번호 1F32GM099374-01 (PDN), 브리검 영 대학교 시동 기금 (JMC, MLK, MV). 출판 비용은 브리검 영 대학교 생명 과학 대학 식물과 야생 동물 과학 부서에 의해 지원되었다.

1. 문화 박테리아 (시작 ~ 1 주 달걀을 따기 전) <ol><li> 수정 MRS (20) (과 MMR) 접시와 국물 튜브 (표 1)을 준비합니다. 각 100mm 페트리 접시에 한천 20 ㎖의 MMRS을 붓고 / 건조 하룻밤을 냉각 할 수 있도록, 또는 5 ML의 MMRS는 18mm 테스트 튜브로 국물. </li><li> 킬 아세토 pomorum, A. tropicalis, 락토 바실러스 브레비스 및 L. 판 한천과 MMR에 plantarum을. 하룻밤 30 ° C에서 아세토을 품어. 밀봉하기 전에 이산화탄소 밀폐 용기와 홍수에 접시를 배치하여 혐기성 유산균을 품어. 하룻밤 30 ° C에서 품어. 참고 : L.를 브레비스 식민지는 24 ~ 48 시간까지 표시되지 않을 수 있습니다. 플레이트를 4 ℃에서 저장 될 수 콜로니 최대 3 주 동안 사용될 수있다. </li><li> 2 ~ 3 일 무균 다이어트 (섹션 5)에 dechorionated 달걀을 전송하기 전에, 테스트 튜브 콘에 MMRS 플레이트에서 하나의 식민지를 선택이닝의 MMRS은 국물. 모두 30 ° C에서, 진탕 아세토 성장과 24 시간 또는 혼탁 할 때까지 정적으로 유산균 성장. </li></ol> 2. 멸균 다이어트를 준비합니다 <ol><li> 2 L의 삼각 플라스크에 멸균 다이어트 (표 2)를 준비한다. 이 3 연속 번 삶은 때까지 음식을 전자 레인지; 각 종기 사이에 섞는다. </li><li> 원뿔 원심 분리기 튜브에 다이어트를 전송하는 동안 교반 유지하기 위해 볶음 접시에 플라스크를 배치합니다. 50 ㎖ 원심 분리기 튜브에 다이어트의 7.5 ml에 전송합니다. 느슨하게 덮여, 오토 클레이브 폴리 프로필렌 랙에 튜브를 모자, 장소. </li><li> 121 ° C에서 오토 클레이브 25 분 15 PSI를 사용하여 비행 다이어트를 소독. 오토 클레이브에서 랙을 제거하고 즉시 냉각 동안 분리하지 않는 다이어트를 위해 수평으로 각 랙을 흔들어. 뚜껑 또는 튜브의 테두리에 다이어트의 전송을 방지하기 위해 수직 선반을 흔들지에주의하십시오. 다이어트 정확히 45 분 동안 진탕 기에서 냉각 할 수 있도록 허용하고다시 손으로 수평으로 다이어트를 흔들. 참고 : 다이어트는 일주일까지에 대해 4-15 ° C에 저장할 수 있습니다. </li><li> 적용 오토 클레이브 랙을 사용하는 대신 (2.1-2.3 단계), 또는 산 방부제를 함유 다이어트에 파리를 높이기 위해 다음 단계를 수행합니다 : <ol><li> 플라스크에 교반 막대와 2 L 플라스크 1 L 액체 음식을 준비합니다. 다이어트를 압력솥. </li><li> 고압 증기 멸균 한 후, 10 ml의 방부제를 추가하고 가열 교반 접시에 계속 저어. 한천은 50 ~ 60 ℃로 냉각되면, 50 ~ 60 ℃에서 가열 플라스크 온도를 바이오 안전성 캐비닛에 가열 교반 판에 플라스크를 이동하고 유지한다. </li><li> 바이오 안전성 캐비닛에서 개별적으로 원뿔 튜브에 피펫 ~ 7.5 ml로. </li></ol></li></ol> 3. 달걀 누워 케이지를 준비합니다 <ol><li> 100 ml의 물, 10g의 맥주 효모, 10g 포도당, 한천 1 g의은 전자 레인지에 의해 포도 주스 한천 플레이트를 확인합니다. 단계 1.1 종기로 3 회를 가져와 냉동 포도 주스 공동의 10g을 추가ncentrate는 한천 접시에 계란의 가시성을 증가시킵니다. </li><li> 한천은 55 ° C로 냉각되면, 100mm 배양 접시에 20 ㎖를 부어 응고 할 수 있습니다. </li><li> 15g의 물에 1g의 맥주 효모를 혼합하여 효모 페이스트와 한천 플레이트의 표면을 커버. 뒤에 얇은 효모 찌꺼기를 떠나, 초과를 부어 다음, 표면이 덮여 확인하는 한천 접시에 효모 페이스트를 따르십시오. 복수의 판을 사용하는 경우, 페이스트를 순차적으로 복수의 플레이트에 붓고 수있다. </li><li> 케이지의 바닥에 한천 플레이트를 전송합니다. 참고 : 32 온스 델리 컨테이너 아크릴 플라이 케이지를위한 좋은 대용품이다. <ol><li> 상단에 구멍을 절단하고 무독성 접착제로 구멍을 통해 통기성 메쉬를 접착하여 32온스 델리 용기 뚜껑을합니다. 접착제이면 수용성 뚜껑 물 노출을 방지한다. </li></ol></li><li> 전송 200-300 컨테이너로 날아와 뚜껑 커버. 을 예방하기 위해 빈 페트리 접시의 뚜껑 메쉬 보호 구멍을 커버원하는 경우 한천 표면에서 t의 증발과 뚜껑 내부에 습기 티슈 페이퍼를 추가합니다. 16 ~ 20 시간 동안 25 ℃로 하룻밤에 파리를 품어. </li></ol> 4. 달걀을 수집 <ol><li> 플라스틱 부싱에 나일론 메쉬를 배치하여 계란 수집 체를 준비합니다. </li><li> 거기에 계란 판을 검색하려면, 빈 용기에 전송하여 케이지에서 파리를 제거합니다. 같은 파리 다음날을 사용할 경우, 새로 yeasted 포도 주스 한천 플레이트를 포함하는 새로운 케이지에 바로 전송할 수 있습니다. 파리 순차 3-5 일 동안 사용될 수있다 </li><li> 한천을 아프게하지주의하면서 깨끗한 페인트 브러시와 한천에서 죽은 파리를 제거합니다. </li><li> 부드럽게, 증류수로 한천 플레이트를 세척 한천 표면에서 계란을 솔질하고, 메쉬를 통해 슬러리를 부어 계란을 수집합니다. 전부 또는 계란의 대부분이 한천 플레이트에서 제거 될 때까지 3 ~ 4 번 반복합니다. </li></ol> 5. Dechorionate 계란과 무균 D로 전송보자구 <ol><li> 70 % 에탄올 (양쪽 포함) 내부를 분사하여 바이오 안전성 캐비닛을 준비합니다. 실험실 조직과 바닥을 닦아, 그리고 ~ 15 분 동안 자외선과 후드를 소독. 에탄올로 분사 즉시 바이오 안전성 캐비닛에 배치하여 모든 비 생물학적 용품 (표본 컵, 붓, 집게, 폐기물 용기, 400 ml의 멸균, 0.6 %의 차아 염소산 나트륨의 100 ml)에 소독. 15 분 동안 UV 광으로 소독. </li><li> 120 ml의 시료 컵 또는 다른 멸균 용기에 계란 부싱을 배치하여이 차아 염소산 나트륨 세척 첫 시작. 단지 천천히 림 아래까지 상기 부싱으로 ~ 0.6 % 차아 염소산 나트륨 용액 90 ㎖에 붓는다. </li><li> 2.5 분 동안 계란을 씻어. 주기적으로 상하 염소산 용액 부싱을 이동 집게를 사용하여 알을 다시 일시. </li><li> 두 번째 시료 컵에 직접 부싱을 전송, 바이오 안전성 캐비닛 내부에, 90 ml의 표백제 사전이 가득. </li><li> 반복바이오 안전성 캐비닛 내부에 5.3 단계. 제 표백 처리의 끝에서, 계란 부싱의 측면에 부착하기 시작한다. </li><li> 수행은 바이오 안전성 캐비닛 5.7-5.8 단계를 반복합니다. </li><li> 표백제를 취소하고 멸균 수로 3 회 부싱을 씻는다. 집게 부싱 이동하여 달걀 각 세정 중에 수회 다시 중단. 세 번째 세척 말 가장 달걀 부싱의 측면에 부착한다. </li><li> 에탄올 살균 붓을 사용하여 멸균 다이어트 부싱 측으로부터 알 옮긴다. 개별적으로 또는 소량의 계란을 전송합니다. 유리 병 당 30 ~ 50 알을 목표로. 산소 튜브를 입력 할 수 있도록 느슨한 모자를 남겨주세요. 유리 병은 무균 남아있는 경우, 곤충 인큐베이터로 전송; 그렇지 않으면, 다음과 같이 박테리아를 추가합니다. </li></ol> 6. 무균 4 세균 종을 사용하여 파리 확인 <ol><li> 박테리아 준비 <ol><li> 필요한 소모품과 살균 바이오 안전성 캐비닛을 준비합니다 (Pipettes, 피펫 팁 상자, 멸균 원심 분리기 튜브, MRS 배지, 단계 4.1에서와 같이 테스트 튜브 랙). 에탄올을 묻힌 연구소와 테스트 튜브의 외부를 닦아은 바이오 안전성 캐비닛에 배치하기 전에 닦아주십시오. </li><li> 제 멸균 미세 원심 분리 튜브로 밤새 성장의 500 μl를 전송하여 세균 펠렛. 세균 밀도가 낮은 경우, 각각의 튜브에 1.5 ml의를 추가 또는 순차적으로 뜨는을 제거하고 동일한 튜브에 여분의 문화를 추가 할 수 있습니다. 10,000 × g으로 10 분 동안 바이오 안전성 캐비닛과 원심 분리기에서 샘플을 제거합니다. 샘플 사이의 오염을 방지하기 위해 필터 팁을 사용합니다. </li><li> OD 600을 측정하여 각 문화의 밀도를 결정합니다. 멀티 - 웰 플레이트 판독기를 사용하는 경우, 1-, 2-, 4- 배 희석 한 96 웰 플레이트에 각 배양 200㎕를 옮긴다. </li><li> 평판 판독 분광기를 사용하여 각 세포 펠렛 (5.2.2) 및 다음 식을 희​​석 할 수있는 MMRS의 양을 결정한다. &amp; 50을 접종하기에 충분한 국물을 추가 할 계획# 956; 각 플라이 유리 병에 리터. <ol><li> 1, 1 : 2, 1 : 판 독서 분광 광도계의 각 세균 배양의 4 희석 OD에게 1 (600) 수치를 수집합니다. 0.1과 0.2이 값과 6.1.4.2 또는 6.1.4.3에 주어진 공식에 &#39;O&#39;와 &#39;D&#39;로 해당 희석 배수를 사용 사이의 OD 600 값을 생성 각 균주의 희석을 선택합니다. </li><li> 여기에 설명 된 4 종을 사용하는 경우, (결정 이전에 CFU 변환에 20 OD 600)이 식을 이용하여 해당하는 집락 형성 단위로 셀 (CFU) / ㎖ 밀도를 정상화 : E = ((OB) × V의 X 용 D) / C E = 볼륨 (μL)에 펠렛을 재현 탁하는 경우, O = OD 600 박테리아, B = OD 600 빈 미디어, D = 배 희석, V = ㎕의 세균 배양 전에 원심 분리, 소정의 상수 C = OD 600. 기업 코드는 다음 방정식을 사용하여 계산 예제 파일을 참조하십시오. 분광 광도계를 들어&quot;OB&quot;대신에 &quot;O&quot;를 자동으로 빈 사용의. 참고 : 다음과 같이 소정의 상수 (10 7 CFU ml를 -1로 정규화 단위 OD (600), (20)에서 파생 된 상수)은 다음과 같습니다 A. tropicalis (0.052), A. pomorum (0.038), L. 브레비스 (0.056), L. 란타 (0.077). </li><li> 다른 박테리아 종을 사용하는 경우,이 방정식을 사용하여 600 = 0.1 중독에 밀도를 정상화 (더 CFU / OD 600 상수는 사용할 수 없습니다) E = ((OB) × V의 X 용 D) /0.1 OD (600) 주 : 단위 6.1.4.2 단계와 동일하다. 기업 코드는 다음 방정식을 사용하여 계산 예제 파일을 참조하십시오. </li></ol></li><li> 바이오 안전성 캐비닛에 피펫 팁으로 뜨는을 제거 단계 6.1.4.2에서 계산 된 신선한 MMRS 또는 PBS의 펠렛을 재현 탁. </li></ol></li><li> 박테리아를 접종 <ol><li> 멸균 다이어트과 원뿔 튜브로 전송 박테리아의 50 μl를D는 바이오 안전성 캐비닛에 알을 dechorionated. 유리 병 사이의 오염을 방지하기 위해 달걀 전송 후 박테리아를 추가합니다. </li><li> 25 ℃에서 인큐베이터에 접종 튜브를 배치 </li></ol></li></ol> 불임 7. 측정 CFU로드 / 테스트 <ol><li> 세라믹 구슬 125 μL와 MMRS 국물의 125 μl를 포함하는 1.7 ml의 미세 원심 분리 튜브에 몸 전체 비행 균질, 전송 5 파리 (5-7일 포스트 우화)의 CFU 부하를 측정합니다. 4.0 M / 초에서 30 초 동안 조직 균질를 사용하여 파리를 균질화. <ol><li> 또한, 구슬과 손을 1 분 동안 플라스틱 방앗와의 microcentrifuge 튜브에 균질화 생략합니다. </li><li> 장내 미생물을 정량화하는 경우, 외래 미생물 (22)을 제거하기 위해 파리를 소독 표면. 전송 homogenizations위한 새로운 마이크로 원심 튜브에 1 분, 흡 에탄올 및 전송을 위해 100 ㎕의 70 % 에탄올을 함유하는 마이크로 원심 튜브에 날아간다. 소화관의 DNA 함량을 측정 할 경우, F 린스또는 에탄올 세척 전에 0.6 %의 차아 염소산 나트륨 1 분. </li></ol></li><li> 875 μL의 MMRS 5 초 동안 소용돌이 균질 희석하고, 마이크로 타이 터 플레이트의 제 1 웰에 120 μL의 파쇄를 피펫. </li><li> 10 μl를 균질하고 다음 두 우물에 70 μl의 MRS를 사용하여 8 희석 :이 순차적를 수행합니다. <ol><li> 첫 번째 우물에서 10 μl를 제거하고 제 2 웰 포함 70 μl를 MRS에 추가합니다. 세 번째도 포함하는 70 μl를 MRS에 전송, 철저하게 제 2 웰에서 10 μl를 두 번째 우물의 내용을 혼합하고, 철저하게 섞는다. 희석하지, 1 : 8, 및 1:64이 3 총 원래 1000 ㎕를 균질의 농도에 연결됩니다. </li></ol></li><li> 전송할 MMRS 플레이트 각 희석액 10 ㎕를 (원하는 경우 멀티 채널 피펫을 사용하여). 약간 한천 표면 아래로 희석을 몇 mm를 확산 액체가 판을 이동하기 전에 건조 할 수 있도록 접시를 경사. 액체는 일에 건조플레이트는 이웃하는 두 방울의 혼합을 감소 2 일 오래된 빠르게 경우 전자 판. </li><li> 1~2일 30 ° C에서 품어. 한 번 별개의 개별 식민지 볼 수 있습니다 인큐베이터에서 접시를 제거하고, 10 ~ 100 절연 식민지와 희석에서 계산합니다. </li><li> 방정식 E = C의 X D / P의 X의 V 비행 당 E는 = CFU, C는 = 콜로니의 수를 계산 / F, D = 희석, P는 μL 플라이 균질의, V는 = 볼륨을 도금 = 사용하여 비행 당 CFU를 계산하고, F = 균질화 파리의 수입니다. </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+genetic+determinants+of+microbiota-dependent+nutrition+revealed+by+genome-wide+analysis+of+Drosophila+melanogaster.">Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster.</a> Nat Commun. 6, 6312 (2015).</li><li>Bakula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+persistence+of+a+microbial+flora+during+postembryogenesis+of+Drosophila+melanogaster.">The persistence of a microbial flora during postembryogenesis of Drosophila melanogaster.</a> J Invertebr Pathol. 14 (3), 365-374 (1969).</li><li>Ryu, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+homeostasis+by+the+homeobox+gene+caudal+and+commensal-gut+mutualism+in+Drosophila.">Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.</a> Science. 319 (5864), 777-782 (2008).</li><li>Blum, J. E., Fischer, C. N., Miles, J., Handelsman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+replenishment+sustains+the+beneficial+microbiome+of+Drosophila+melanogaster.">Frequent replenishment sustains the beneficial microbiome of Drosophila melanogaster.</a> MBio. 4 (6), 00860 (2013).</li><li>Bitner-Mathe, B. C., Klaczko, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Drosophila+melanogaster+wing+morphology:+effects+of+sex,+temperature+and+density.">Plasticity of Drosophila melanogaster wing morphology: effects of sex, temperature and density.</a> Genetica. 105 (2), 203-210 (1999).</li><li>Edward, D. A., Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+effects+of+developmental+environment+on+reproductive+trait+expression+in+Drosophila+melanogaster.">Sex-specific effects of developmental environment on reproductive trait expression in Drosophila melanogaster.</a> Ecol Evol. 2 (7), 1362-1370 (2012).</li><li>Ridley, E. V., Wong, A. C., Westmiller, S., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+resident+microbiota+on+the+nutritional+phenotype+of+Drosophila+melanogaster.">Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster.</a> PLoS One. 7 (5), e36765 (2012).</li><li>Newell, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+function+and+comparative+genomic+analyses+of+the+Drosophila+gut+microbiota+identify+candidate+symbiosis+factors.">In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors.</a> Front Microbiol. 5, 576 (2014).</li><li>Dewhirst, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+the+defined+murine+microbiota:+altered+Schaedler+flora.">Phylogeny of the defined murine microbiota: altered Schaedler flora.</a> Appl Environ Microbiol. 65 (8), 3287-3292 (1999).</li><li>Min, K. T., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia,+normally+a+symbiont+of+Drosophila,+can+be+virulent,+causing+degeneration+and+early+death.">Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death.</a> Proc Natl Acad Sci U S A. 94 (20), 10792-10796 (1997).</li></ol>

저자는 공개 아무것도 없어.

<html> 여기에 설명 된 방법은 함께 무균 성인 18,27의 시리얼 전송 또는 항생제 치료 13,18 포함 무균 파리, 양육의 대체 방법으로, 배아 dechorionation 8,11,18,25,26,27에 대한 몇 가지 방법 중 하나입니다. 다른 dechorionation 방법은 에탄올 세척을 포함 11,25,26를 줄이거 나 8 차아 염소산 치료를 확장합니다. (에탄올 세척하지 않고) 여기에 설명 된대로 사육 할 때 100 ?...

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.
Step 6.1.4.2 of the Protocol was updated from:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.052), A. pomorum (0.038), L. brevis (0.056), L. plantarum (0.077).
to:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.053), A. pomorum (0.038), L. brevis (0.077), L. plantarum (0.056).

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.

Erratum: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

대부분의 동물은 밀접하게 죽음 일에 출생에서 박테리아 ( &#39;미생물&#39;)과 연결되어 있습니다. 미생물이없는 ( &#39;무균&#39;) 및 미생물 관련 ( &#39;기존&#39;) 동물의 비교는 미생물 대사, 영양, 혈관, 간, 호흡기, 면역, 내분비 및 신경 기능이 포함 동물 건강의 다양한 측면을 영향을 보여 주었다. 열매는 초파리 melanogaster의 3,4- 미생물의 존재 하에서 이러한 프로세스의 많은 이해 및 동물 건강에 5,6- 미생물의 영향을 연구하기위한 주요 모델 비행. 어떤 박테리아 종은 모든 개인 ( &#39;코어&#39;)에 존재하지 않지만 아세토 및 락토 바실러스 종은 수치 모두 실험실 사육 및 야생 잡은 D.의 미생물을 지배 melanogaster의. Acetobacteraceae, Firmi (Komagataeibacter 및 글루 포함) 기타및 장내 세균 (예 : 엔테로 및 로이코 노 스톡 등) cutes 중 하나 높은 풍부 7-12에서 낮은 풍부에서 초파리 개인에서 자주 본, 또는 불규칙하게 존재한다. 초파리와 포유 동물의 미생물은 내 세대 14,19에 걸쳐 일정하지 않습니다. 미생물에 의존하는 특성을 측정 할 때 미생물의 변덕은 표현형 노이즈가 발생할 수 있습니다. 예를 들어, 초파리 15-18에서 Acetobacteraceae 영향 지질 (중성 지방) 저장. Acetobacteraceae 다른 19 이상의 유리 병의 파리에서 더 풍부 경우, 동종 파리는 서로 다른 표현형 (20)를 가질 수있다. 쥐 14 미생물의 변덕의 문제에 대한 해결책은 마우스 새끼로 (Schaedler 식물 변경) 각각의 새로운 세대를 8 지배적 인 미생물 종의 정의 사회를 도입하여, 1960 년대 이후 실제로있다각 강아지가 마우스 미생물 동일한 키 부재에 노출되는 것을 보장한다. 미생물이 연구 (32)의 주 대상이 아니며, 실험 조건의 다양한 키 미생물의 존재를 확인하기 위해 전례를 설정하는 경우에도이 방법은 미생물 조성에 대한 제어합니다. 초파리 영양 미생물의 영향을 정의하려면, 무균 플라이 라인을 도출하기위한 여러 가지 프로토콜은 배아의 차아 염소산 dechorionation (중 파생 드 노보 각 세대 또는 멸균 사료로 전송하여 generationally 유지) 및 항생제 치료 (13)를 포함하여 개발되었다. 항생제 처리 및 시리얼 전송, 드 노보 dechorionation와 교란 변수 (예를 들어, 계란 밀도, 잔류 오염 미생물, 오프 - 타겟 항생제 효과)의 대 더 제어 모두 용이성과 신속성 등 다양한 접근 방법에 장점이있다. 상관없이 방법의배아를 무균하는 특정 미생물 종의 준비, 도입은 정의 ( &#39;무균&#39;) 사회와 초파리의 문화를 허용한다. 또한, Schaedler 식물의 사용을 모방,이 커뮤니티는 각 유리 병의 특성-에 영향을 미치는 미생물의 존재를 확인하고 미생물의 변덕의 합병증을 피하기 위해 (만 단계 6-7 다음) 종래 누워 계란에 접종 할 수있다. 여기에서 우리는 배아의 드 노보 dechorionation에 의해 무균 및 무균 초파리를 제기하기위한 프로토콜을 설명하고 소개 또는 오염 미생물 분류군의 존재를 확인합니다.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Brewer&#39;s Yeast</td>
			<td style="width:291px;">MP Biomedicals, LLC.</td>
			<td style="width:108px;">903312</td>
			<td style="width:195px;">http://www.mpbio.com/product.php?pid=02903312</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma Aldrich</td>
			<td>158968-3KG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en&#38;region=US</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Fisher--Lab Scientific</td>
			<td>fly802010</td>
			<td>https://www.fishersci.com/shop/products/drosophila-agar-8-100mesh-10kg/nc9349177</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Welch&#39;s 100% Grape Juice Concentrate</td>
			<td>Walmart or other grocery store</td>
			<td>9116196</td>
			<td>http://www.walmart.com/ip/Welch-s-Frozen-100-Grape-Juice-Concentrate-11.5-oz/10804406</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cage: 32 oz. Translucent Round Deli Container</td>
			<td>Webstaurant Store</td>
			<td>999L5032Y</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-sd5032y-32-oz-translucent-round-deli-container-24-pack/999L5032Y.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Translucent Round Deli Container Lid</td>
			<td>Webstaurant Store</td>
			<td>999YNL500</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-ynl500-translucent-round-deli-container-lid-60-pack/999YNL500.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stock Bottles</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">32-130</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=32-130</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Droso-Plugs</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">49-101</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=49-101</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nylon Mesh</td>
			<td>Genesee Scientific</td>
			<td>57-102&#160;</td>
			<td>https://geneseesci.com/shop-online/product-details/715/?product=57-102</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Bushing</td>
			<td>Home Depot</td>
			<td style="width:108px;">100343125</td>
			<td>http://www.homedepot.com/p/Halex-2-1-2-in-Rigid-Insulated-Plastic-Bushing-75225/100343125</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specimen Cup</td>
			<td>MedSupply Partners</td>
			<td>K01-207067</td>
			<td>http://www.medsupplypartners.com/covidien-specimen-containers.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Repeater M4</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>https://online-shop.eppendorf.us/US-en/Manual-Liquid-Handling-44563/Dispensers--Burettes-44566/Repeater-M4-PF-44619.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml Centrifuge Tubes</td>
			<td>Laboratory Product Sales</td>
			<td>TR2003</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Food Boxes</td>
			<td>USA Scientific</td>
			<td>2316-5001</td>
			<td>http://www.usascientific.com/search.aspx?find=2316-5001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Matrix D Bulk</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116540434</td>
			<td>http://www.mpbio.com/search.php?q=6540-434&#38;s=Search</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 300&#956;l</td>
			<td>USA Scientific</td>
			<td>1120-9810</td>
			<td>http://www.usascientific.com/search.aspx?find=1120-9810</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Laboratory Product Sales</td>
			<td>M089303</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Decon Laboratories, INC.</td>
			<td>2701</td>
			<td>http://www.deconlabs.com/products.php?ID=88</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paintbrush</td>
			<td>Walmart</td>
			<td>5133</td>
			<td>http://www.walmart.com/ip/Chenille-Kraft-5133-Acrylic-Handled-Brush-Set-Assorted-Sizes-colors-8-Brushes-set/41446005</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Fisher</td>
			<td>08-882</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-medium-pointed-forceps-3/p-128693</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Household Bleach (6-8% Hypochlorite)</td>
			<td>Walmart</td>
			<td>550646751</td>
			<td>http://www.walmart.com/ip/Clorox-Concentrated-Regular-Bleach-121-fl-oz/21618295</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Universal Peptone</td>
			<td>Genesee Scientific</td>
			<td>20-260</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=20-260</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast Extract</td>
			<td>&#160;Fisher Scientific</td>
			<td>BP1422-500</td>
			<td>https://www.fishersci.com/shop/products/fisher-bioreagents-microbiology-media-additives-yeast-extract-3/bp1422500?matchedCatNo=BP1422500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dipotassium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>P3786-1KG</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Citrate</td>
			<td>Sigma Aldrich</td>
			<td>25102-500g</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=25102-500g&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Acetate</td>
			<td>VWR</td>
			<td>97061-994</td>
			<td>https://us.vwr.com/store/catalog/product.jsp?catalog_number=97061-994</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?matchedCatNo=M63500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manganese Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>10034-96-5</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=10034-96-5&#38;interface=CAS%20No.&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MRS Powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:108px;">69966-500G</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en&#38;region=US</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plate Reader</td>
			<td>BioTek (Epoch)&#160;</td>
			<td>NA</td>
			<td>http://www.biotek.com/products/microplate_detection/epoch_microplate_spectrophotometer.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.7 ml Centrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>http://www.usascientific.com/search.aspx?find=1615-5500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 1000&#956;l</td>
			<td>USA Scientific</td>
			<td>1122-1830</td>
			<td>http://www.usascientific.com/search.aspx?find=1122-1830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plates</td>
			<td>Greiner Bio-One</td>
			<td>655101</td>
			<td>https://shop.gbo.com/en/usa/articles/catalogue/article/0110_0040_0120_0010/13243/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ceramic Beads</td>
			<td>MP Biomedicals, LLC.</td>
			<td>6540-434</td>
			<td>http://www.mpbio.com/product.php?pid=116540434</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Homogenizer</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116004500</td>
			<td>http://www.mpbio.com/product.php?pid=116004500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Class 1 BioSafety Cabinet</td>
			<td>Thermo Scientific&#160;</td>
			<td>Model 1395</td>
			<td>http://www.thermoscientific.com/en/product/1300-series-class-ii-type-a2-biological-safety-cabinet-packages.html</td>
		</tr>
	</tbody>
</table>

rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

무균 파리의 성공적인 양육은 D.의 전신 homogenizations에서 더 CFUs의 분리에 의해 확인 melanogaster의 성인 (그림 1). 도금 균질가 식민지를 산출하는 경우 또는, 유리 병은 오염 및 폐기해야합니다. 무균 파리를 들어, 네 개의 박테리아 균주의 각은 성인 파리 (그림 1)과 관련된 총 가능한 CFUs의 차이를 보여, 5 성인 남성의 풀에서 분리 하였?...

Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

무균 및 무균 조건에서 초파리 melanogaster의를 양육하는 방법이 제공된다. 플라이 배아는, 차아 염소산 나트륨에 dechorionated 멸균 다이어트에 무균 적으로 전송하고, 밀폐 용기에 양육된다. 박테리아와식이 요법과 배아를 접종은 무균 협회에 이르게하고, 세균의 존재는 전신 초파리 균질 도금에 의해 확인된다.

과일 비행을 양육<em&gt; 노랑 초파리</em&gt; 무균 및 무균 조건에서

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic

Research

JoVE Journal

Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

21.0K 조회수.  Brigham Young University.  무균 및 무균 조건에서 초파리 melanogaster의를 양육하는 방법이 제공된다. 플라이 배아는, 차아 염소산 나트륨에 dechorionated 멸균 다이어트에 무균 적으로 전송하고, 밀폐 용기에 양육된다. 박테리아와식이 요법과 배아를 접종은 무균 협회에 이르게하고, 세균의 존재는 전신 초파리 균질 도금에 의해 확인된다. 

비디오: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

의 시금 혈액 세포 집단<em&gt; 초파리</em&gt; 유충

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

연구

발생학

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

양육 먼저 먹이 제브라 피쉬의 전체 및 업데이트 &quot;로티퍼 다종 양식 방법&quot;

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

제노없는 조건에서 3-D 문화의 생산과 치료 중간 엽 줄기의 관리 / 기질 세포 (MSC) 타원체 뇌관

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

과일 플라이의 면역 염색과 Steroidogenic 기관 및 그들의 상호 작용하는 장기를 시각화하기위한 프로토콜<em&gt; 노랑 초파리</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

낮은 혈 청 조건 하에서 심장 조상 세포에서 내 피 분화의 유도

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

녹고, 경작, 그리고 초파리 셀 라인 Cryopreserving

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

초파리 멜라노가스터에서 내분비 장애를 테스트하는 방법

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

초파리 멜라노가스터의 혈액-뇌 장벽 무결성에 대한 분석

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

피더가 없고 화학적으로 정의된 배양 조건 하에서 인간 다능성 줄기 세포를 사용하여 후갱이온 교감 뉴런의 효율적인 분화

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

과일 비행을 양육

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양육 먼저 먹이 제브라 피쉬의 전체 및 업데이트 "로티퍼 다종 양식 방법"