환경 보호 및 연구를위한 연구소 Loredana Manfra (환경 품질 모니터링의 ISPRA -학과) 및 환경 보호를위한 지역기구 (에밀리아 - Romagna, 이탈리아)의 Federica Savorelli에 의해 제작. 바닷물 / 소금기있는 물 및 UNICHIM위원회 수질에 (화학 분야의 기술 표준화 운영을위한 연구원)의 침전 - 생물 학적 방법 그룹 내에서 Artemia 갑각류의 서브 작업 그룹에 대한 참조 멤버. ISPRA의 마르코 Pisapia (웹 단위)에 의해 Videography 및 편집. 안나 마리아 시세 및 ISPRA의 에리카 Magaletti (환경 품질 모니터링학과)에 의한 생산 조정. 바닷물 / 소금기있는 - 우리는 에밀리아 - Romagna의 환경 보호, 페라라 지점, 생물 학적 방법 그룹에 대해 지역 기관에게 감사를 표합니다Rossella Boscolo과 재정 지원을위한 ISPRA의 마시모 Gabellini (예방과 영향의 완화의학과), 파비오 Matassa와 런던 수질 및 Luciana Migliore, 그들의 협력 토르 Vergata 대학 (로마)에서 UNICHIM위원회 물과 침전물 그들의 언어에 대한 지원 로마의 언어 학원.

1. 방법 표준 우리의 테스트는 생물의 50 %의 사망 (LC 50) 14 일 동안 노출이 발생할 것입 농도 또는 희석 수준을 결정하는 목적으로 약물 농도의 간격이나 테스트 샘플에 Artemia의 애벌레를 노출하고 정의된 조건을 충족 포함 이 방법에 의해. 필요하고 가능하다면, 다음도 결정될 수 있습니다) 노출 생물의 20 % (LC 20)의 사망 원인이 농도 수준, B)보다 높은 사망률을 결정하지 않는 최고의 분석 농도 수준 부정적인 컨트롤 (NOEC); 십사일, 부정적인 컨트롤 (LOEC)보다 높은 사망률 후 결정 낮은 테스트 농도 수준. 2. 테스트 재료 2.1 Artemia 달걀 테스트를 수행하려면 Artemia franciscana </em옛 미만 48 시간&gt; 애벌레는 (nauplii) 새로 부화 계란 5에서 얻은 무대 II 또는 III에서 필요합니다. 테스트를 지원하기 위해 활용 공인 계란이나 cysts는 품질 보증 연구 부문, 미국 환경 보호청, 신시내티, 오하이오 45268 미국이나 수생 오염의 생물 학적 연구, 겐트 대학, 벨기에의 연구실에서 예를 들어 상업적으로 사용할 수 있습니다. 2.2 물의 희석 합성 바닷물이 인식 분석 품질 시약 또는 증류수 또는 드 이온화 물에 시판 제제를 해소하여 준비 희석 물로 사용할 수 있습니다. 이상적인 바닷물을 만들기위한 소금 조화가 상업적으로 사용할 수 인스턴트 오션 섞어 예. 그것은 35 (± 2)와 동일한 염도로 희석 물을 권장합니다 PSU뿐만 아니라 채도의 가치의 80 % 이상의 용존산소 농도 및 25의 온도 (± 2) &amp;있길; C. 48시간 적어도 0.45 μm의의 다공성과 필터의 여과를위한 통기 후 희석은 0-4 ° C의 온도에서 어둠 속에서 30 일 최대 저장될 수 있습니다 2.3 Algal 문화 2.0x10 6 셀 / ML - 이것은 시험 생물은 기하 급수적으로 성장하고 1.3x10 6 밀도에 도달 Dunaliella tertiolecta의 microalgae에 피드입니다 것을 권장합니다. 환경 시료를 테스트하는 것은 2.0x10 6 셀 / ML 6보다 높은 밀도와 문화가 필요합니다. 이는 Dunaliella tertiolecta는 해수가 들어있는 문화 매체에서 자랍니다 것이 좋습니다 (30, (± 2) PSU 20 (± 1) ℃) 필수 영양소, 미네랄과 비타민의 추가와. algal 미디어 조성과 준비에 대한 일부 징후는 부록에보고됩니다. 초기 세포 밀도는 약 10 5 세포 / ML로 추천됩니다. algal 문화는 유지되어20 객실 온도 조절기 (± 1) ° C, 어둠의 8 시간 빛의 16 시간 photoperiod 비율과 형광등 3000 (± 300) LX와 조명. 기타 식물성 플랑크톤 종은 또한 먹이를 위해 사용될 수 있지만 Dunaliella tertiolecta에 &#39;동등한&#39;종을 찾는 이들 식물성 플랑크톤 종과 비교 연구를 할 필요가있을 것이다 때문에 실험 조건 (예 : 초기 세포 밀도, 라이트 조명, 온도의 구성 문화 미디어와 노출 시간)은 식물성 플랑크톤 종 중 다릅니다. 다른 오염 물질에 대한 microalgae의 공차가 고려되어야 외에 때문에 또한 Artemia의 식물성 플랑크톤에 미치는 영향의 성장과 발전에 대한 오염 물질 영향. 2.4 참조 물질 이 테스트에서 기준 물질은 나트륨 Dodecyl 황산이다. 2.5 실험실 유리 제품 <ol><li> 100 ML borosilicate 유리 비커 (18 솔루션을 전송하는 시험과 18 수행)를 포함하여 테스트 컨테이너. </li><li> 표준 용액을 준비하는 플라스크 (예 : 500 ML 플라스크). </li><li> 테스트 솔루션을 준비하는 식스 flasks (예 : 200 ML의 flasks). </li><li> 이동식있는 유리 또는 폴리스티렌 5cm 직경 배양 접시는 cysts를 활성화하고 테스트 컨테이너에 부화 요리에서 Artemia를 전송에 사용되는 다룹니다. </li><li> nauplii를 전송하는 유리 파스퇴르의 pipettes (불꽃에 의해 둥근 팁 포함). </li><li> 유리 캐뉼러 또는 Artemia를 전송하는 파스퇴르 3 ML 일회용 플라스틱 pipettes (절단되어 있어야합니다.) </li><li> Micropipettes 및 단계적 pipettes은 테스트 솔루션을 준비합니다. </li><li> t로 flasks에서 테스트하는 솔루션을 전송하기 위해 6 단계적 실린더 (예 : 50 ML 실린더)그는 용기를 시험. </li></ol> 3. 낭종 활성화 3.1. cysts가 들어있는 배양 접시 +는 물을 붓는 테스트 유기체를 생성하려면, cysts 20 밀리그램은 48 시간 테스트하기 전에 희석 물 12 ML을 포함하는 배양 접시에 넣습니다. 3.2. 빛과 어둠 속에서 부화 배양 접시가 (± 2) 25에서 관리하고 있습니다 ° C와 4000 (± 1000) 럭스 (평방 미터 당 루멘) 1 시간 빛의 강도에. 3.3. 현미경을 사용하여 매체를 교체 24 시간 이내에, 부화 유충은 인공 물이 담겨있는 새로운 배양 접시로 전송됩니다. 송금은 부화, phototropic 애벌레는 광선으로 마이그레이션할 수 있도록 즉, 광원을 사용하여, 현미경을 사용하여 수행됩니다. 유리 파스퇴르 피펫은 그만이 초기 유충 AR을 보장, 전송을 확인하는 데 사용됩니다E는 세포막 아직 cysts이나 애벌레를 양도 및 없습니다. 3.4. 어둠 속에서 부화 애벌레가 들어있는 요리는 25 24 시간 동안 어두운 온도 조절 챔버에 배치됩니다 (± 2) ° C. 4. 테스트 솔루션의 작성 4.1. 물질을 계량하는 것은 + 플라스크에 양도 이것은 시험 물질의 표준 솔루션 500 ML 플라스크에서 물질의 0.5 g을 해소하여 준비하는 것이 좋습니다. 플라스크는 드 이온화 또는 증류수로 가득 차 있으며 시험 물질이 완전히 해소되기 전까지 솔루션은 흔들됩니다. 솔루션은 그것이 물질이 용액에 안정 여부를 알 수 없을 경우를 제외하고 사용 시간에 대비해야합니다. 이 경우, 표준 솔루션은 테스트를 떠나기 2 일 이내에 준비됩니다. 4.2. 희석 물 조류 WI를 추가, 부정적인 제어를 준비10 ML 피펫 일 부정적인 컨트롤은 10 5 세포 / ML의 밀도가 얻어진다되도록 희석 물에 algal 정지 나누어지는를 추가하여 플라스크 (예 : 200 ML 볼륨)에 준비가되어 있습니다. 4.3. 테스트 솔루션을 준비 : 희석 물, 조류 및 표준 용액을 추가 이것은 테스트 솔루션 테스트 원하는 농도가 취득되도록 지정 수량의 희석 물에 표준 용액을 추가하여 최대 5 개의 200 ML의 flasks에 준​​비가되어 있다고 제안한다. 생물 먹이려면 Dunaliella tertiolecta의 microalgae 서스펜션의 aliquots 사용 및 테스트 솔루션이 준비되면 10 5 세포 / ML의 밀도에 도달 추가될 수 있습니다. 이외의 다음과 같은 순서를 권장합니다 : 희석 물, algal 정지 및 표준 용액을. 테스트 솔루션의 준비 후, 그들은 가능​​한 한 빨리 분석에 사용해야합니다. 4.4. 후 배양 접시에, 플라스크에서 테스트 컨테이너에 대한 솔루션을 전송하기 테스트 솔루션의 평등 개당 (50 ML)를 졸업 실린더를 이용한 테스트 컨테이너에 소개됩니다. 각 농도에 대한 세 복제본이 만들어집니다. 시험의 각 시리즈의 경우 컨트롤 컨테이너는 테스트 솔루션의 부피와 동일한 희석 물을 금액으로 준비가되어 있습니다. 테스트 솔루션의 동등 권 (12 ML)는 배양 접시에 소개됩니다. 시험의 각 시리즈 들어, 컨트롤 배양 접시가 사용됩니다. 5. 테스트를위한 준비 5.1. II-III 단계에서 Nauplii 낭종 인증 후 마흔 8 시간, nauplii는 II-III 애벌레 단계에 도달하고 테스트 후 사용할 수 있습니다. 5.2. 현미경을 사용하여 배양 접시에 Nauplii 전송 낭종에 사용된 배양 접시<html> 정품 인증은 온도 조절 챔버 밖으로 가져옵니다. 애벌레의 작은 수량은 제어 및 테스트 솔루션을 포함하는 배양 접시로 전송됩니다. 이 작업은 유기체가 손상되지 않도록 충분히 넓은 직경의 피펫을 이용한 현미경으로 수행되어야하는 것입니다. 시험이 단계에서는 전송이 유리 파스퇴르 피펫 (불꽃에 의해 둥근 끝)와 집계에 Artemia을 장려하는 laterally 위치를 광원으로 수행할 수 있습니다. 5.3. 현미경으로 테스트 컨테이너에 페트리 접시에서 Nauplii 전송 열 유충은 테스트 컨테이너에 테스트 솔루션을 배양 접시로부터 전송됩니다. 또한이 작업은 파스퇴르 피펫과 현미경을 이용하여 수행되어야한다. 그것은 한 ML을 초과하지 볼륨 테스트 시스템의 전체적인 볼륨에 영향을 미치지 않도록 애벌레의 흐름 동안 전송되는 것을 권장합니다. e_step &quot;&gt; 5.4. 전체 컨테이너 parafilm 덮여 테스트 비커는 parafilm (공기 통로에 대한 간격을두고)으로 덮여, 25의 온도 (± 2) ° C 시험 전체 기간 동안 900 (± 100) LX의 조명에서 photoperiod과에서 관리하고 있습니다 어둠의 10 시간 빛의 14 시간 비율입니다. 6. 중형과 음식 보충 교체 6.1. 서바이벌 속도 제어 테스트가 시작된 이틀 후, 그리고 5, 7, 9, 12 일 후에, Artemia가 생존 속도를 확인하고 매체 및 식품 보충제를 대체하는 현미경으로 관찰한다. 살아있는 생물의 개수는 각 테스트 컨테이너에 계산됩니다. 현미경으로 관찰하고 약간의 기계적 자극 (예 : 유리 파스퇴르 피펫과 애벌레를 만지고) 10의 몇 가지 움직임을 보여주지 않는 생물 후econds 죽은 것으로 간주되어야한다. 6.2. micropipette으로 테스트할 수있는 물질의 10 ML의 피펫과 샘플링과 조류 샘플링을 보여주는 테스트 솔루션의 준비. 플라스크에서 테스트 컨테이너에 대한 해결책을 전송하기 테스트하는 동안 테스트 솔루션은 주기적으로 교체하고, 그들이 사용할 수있는대로 같은 날 준비를해야합니다. 시험 솔루션은 이전에 준비한 표준 용액으로 만들어집니다. 세 개의 새로운 용기로 이전 테스트 컨테이너에서 6.3. Artemia 전송, 잘라내기 파스퇴르 피펫을 활용, Artemia의 양도 너무과 같은 유기체를 손상하지 않도록 충분히 넓은 직경과 피펫을 사용하여 수행됩니다. 이 단계 동안 3 ML 플라스틱 파스퇴르 피펫은 (절단 예정) 사용할 수 있습니다. 6.4. 어느 라이브 또는 죽은 Artemia을 포함 비커에 애벌레 1 일 이후<html> 테스트의 마지막에 노출된 4 일, 유충에도 살아날 수는 계산하고 워크시트에 기록됩니다. LC 50 및 / 또는 LC 20 NOEC 14 일에서 LOEC는 계산하고 기록뿐만 아니라 자신감을 제한 95% 적절한, 그리고 계산 방법에서. 7. 대표 결과 시험 (14 일)의 끝에 다음 공식에 따라 각 농도에서 사망률의 비율을 계산 : (M S / N) * 100 장소 : M S는 분석 표본에서 죽은 개인의 평균 수입니다 N은 노출된 개인의 총계입니다 표 1 보고서 나트륨 Dodecyl 황산에 노출 Artemia과 테스트 (14d)의 데이터 예제를. <table border="1"><tbody><tr><td> 농도 (MG / L) </td>&quot;각 복제에서 죽은 개인의&gt; 번호 </td><td> 사망률 (%) </td></tr><tr><td></td><td> 나 </td><td> II </td><td> III </td><td></td></tr><tr><td> 음성 제어 </td><td> 0 </td><td> 2 </td><td> 일 </td><td> 10 </td></tr><tr><td> 1.56 </td><td> 일 </td><td> 2 </td><td> 0 </td><td> 10 </td></tr><tr><td> 3.12 </td><td> 2 </td><td> 0 </td><td> 2 </td><td> 13 </td></tr><tr><td> 6.25 </td><td> 3 </td><td> 2 </td><td> 3 </td><td> 27 </td></tr><tr><td> 12.5 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 90 </td></tr><tr><td> 25.0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr><tr><td> 50.0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr></tbody></table> 더욱이을 결정그래픽 가우스 로그 플롯이나 적절한 통계 방법 (예 : 창병-Karber이나 Probit 방법)을 사용하여 LC 50 값. 테이블 1의 데이터에 대한 LC 50 계산은 8.18 (7.15-9.37) MG / L입니다. 최대 농도가 50 %의 낮은 사망률의 원인을 검사하면 다음 신뢰할 심지어 비 계산할 수있을 것이다 LC 50 값의 계산으로 진행해서는 안됩니다. 이 경우 단, 시험 농도의 범위를 확장하여 테스트를 반복하는 적절한 것입니다. 또는 LC 50 값이 더 정확하다면 검사 높은 농도에서 사망률의 비율과 효과 없음에 대응하는 가장 높은 농도를 나타내는 검사 높은 농도보다 클으로 표시됩니다. 다음 조건을 테스트 끝에 충족되었는지 결과는 유효한 것으로 간주됩니다 : <ol class="lalpha"><li> 컨트롤의 평균 사망률≤ 20 %이다; </li><li> 십사일에 나트륨 Dodecyl 황산, LC 50 사용은 8.0 (± 5) MG / L 간격에 포함되는 위치. </li></ol> 위의 조건이 충족되지 않은 경우, 생물의 동일한 배치로 얻은 모든 데이터는 무효로 간주하고 테스트를 반복해야합니다.

<ol>
	<li>Togulga, 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+Short-Term+Toxicity+of+Two+Toxicants+to+Artemia+Nauplii.">The Short-Term Toxicity of Two Toxicants to Artemia Nauplii.</a> Tr. J. of Zoology. 22, 259-266 (1998).</li><li>Persoone, G., Wells, P. G., Sorgeloos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+in+aquatic+toxicology:+a+review.">Artemia in aquatic toxicology: a review.</a> Artemia Research and its Applications. Morphology, Genetics, Strain characterization Toxicology. , 259-275 (1987).</li><li>Nunes, B. S., Carvalho, F. D., Guilhermino, L. M., Stappen, G. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+genus+Artemia+in+ecotoxicity+testing.">Use of the genus Artemia in ecotoxicity testing.</a> Envir. Poll. 144, 453-462 (2006).</li><li>APAT IRSA-CNR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metodi analitici per le acque Manuali e Linee guida 29/2003. Terzo Metodo, 8060 (2003).</li><li>Sorgellos, P., Wielen, C. R. e. m. i. c. h. e. -. V. a. n. D. e. r., Persoone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Artemia+nauplii+for+toxicity+tests+â€“+a+critical+analysis.">The use of Artemia nauplii for toxicity tests â€“ a critical analysis.</a> Ecotox. Env. Saf. 2, 249-255 (1978).</li><li>Savorelli, F., Palazzi, D., Gorbi, G., Invidia, M., Sei, S., Magaletti, E., Manfra, L., Gelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Set+up+of+a+standard+methodology+for+14-day+bioassay+on+Artemia+franciscana+and+A.+parthenogenetica.">Set up of a standard methodology for 14-day bioassay on Artemia franciscana and A. parthenogenetica.</a> Biol. Amb. 21, 27-36 (2007).</li><li>Brix, K. V., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+toxicity+of+arsenic+to+the+Great+Salt+Lake+brine+shrimp,+Artemia+franciscana.">Chronic toxicity of arsenic to the Great Salt Lake brine shrimp, Artemia franciscana.</a> Ecotox. Environ. Saf. 54, 169-175 (2003).</li><li>Brix, K. V., Deforest, D. K., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+chronic+site-specific+water+quality+standard+for+selenium+in+the+Great+Salt+Lake,+Utah,+USA.">Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA.</a> Environ. Toxicol. Chem. 23, 606-612 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualit&#225;+dell'acqua+-+Determinazione+della+tossicit&#225;+letale+a+14+giorni+con+Artemia+franciscana+(Crustacea:+Anostraca).">Qualit&#225; dell'acqua - Determinazione della tossicit&#225; letale a 14 giorni con Artemia franciscana (Crustacea: Anostraca).</a> Commissione UNICHIM "Qualit&#225; dell'acqua" Gruppo di Lavoro "Metodi Biologici" Sottogruppo "Acque salate/salmastre e sedimenti". , (2010).</li><li>Varo, I., Navarro, J. C., Amat, F., Guilhermino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cholinesterases+and+evaluation+of+the+inhibitory+potential+of+chlorpyrifos+and+dichlorvos+to+Artemia+salina+and+Artemia+parthenogenetica.">Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica.</a> Chem. 48, 563-569 (2002).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

Artemia 우리는 독극물과 Ecotoxicology의 Artemia 사용과 관련된 몇 가지 옵션을 유지하는 것이 가능하다고 명시 수 있도록 지금까지 한 ecotoxicity 테스트 및 연구에 사용할 수있는 가장 가치있는 시험 생물 중 하나입니다. bioassays에 적합한 수종에이 유기체를 돌려 특징은 다음과 같습니다 넓은 지리적 분포, 불리한 환경 조건과 다양한 영양소, 비교적 간단한 실험실 문화 유지 보수, 조작에 ?...

환경 보호 및 연구를위한 연구소 Loredana Manfra (환경 품질 모니터링의 ISPRA -학과) 및 환경 보호를위한 지역기구 (에밀리아 - Romagna, 이탈리아)의 Federica Savorelli에 의해 제작. 바닷물 / 소금기있는 물 및 UNICHIM위원회 수질에 (화학 분야의 기술 표준화 운영을위한 연구원)의 침전 - 생물 학적 방법 그룹 내에서 Artemia 갑각류의 서브 작업 그룹에 대한 참조 멤버. ISPRA (과학 자문 및 문서 서비스)의 마르코 Pisapia 의한 Videography 및 편집. 안나 마리아 시세 및 ISPRA의 에리카 Magaletti (환경 품질 모니터링학과)에 의한 생산 조정. 우리는 에밀리아 - Romagna의 환경 보호를 위해 지역 기관에게 감사 페라라 지점과 Biolog을 사고 싶은데요ICAL 방법 그룹 - 바닷물 / 소금기있는 물과 협력 수질에 UNICHIM위원회 침전, Rossella Boscolo과 재정 지원을위한 ISPRA의 마시모 Gabellini (예방과 영향의 완화의학과), 그리고 로마의 언어의 런던 학교 그들의 언어적 지원. <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 제품 번호 </td></tr><tr><td> 나트륨 Dodecyl 황산 </td><td> 시그마 - 올드 리치 </td><td> L4390 </td></tr></tbody></table> 충수 algal 미디어 분석 품질의 시약을 사용하여 대비해야한다. 영양 용액의 리터는 나노 3, 증류수의 NH 4 망할 CIA의 KH 2 PO 4와 20g의 30g 300 G를 해소하여 준비가되어 있습니다. 포 초기 솔루션 (A, B, C, D미네랄)은 다음과 같이 준비가되어 있습니다 : <ul><li> 솔루션의 리터 &quot;&quot;CuSO 4 ZnSO 4 H 2 O, 25g 5 H 2 O, CoSO 4 7 H 2 O, 30 G와 증류수의 MnSO 4 H 2 O의 20g 중 30g이 포함된; </li><li> 솔루션의 리터 증류수에 FeCl 3 • 6 H 2 O, 50 G를 포함하는 &quot;B&quot;; </li><li> 솔루션의 리터 증류수에있는 나 2 무 4 • 2 H 2 O의 25 G를 포함하는 &quot;C&quot;; </li><li> 솔루션을 증류수에 50g 디 나이 EDTA (에틸렌 다이아 민 테트라 초산) • 2 H 2 O를 포함하는 &quot;D&quot;의 리터. </li></ul> 미네랄 (솔루션 &#39;ㅇ&#39;)의 최종 솔루션의 리터는 증류수로 위에서 언급한 솔루션 aliquots를 추가 얻어진다. 이외의 다음과 같은 순서를 권장합니다 : 솔루션 &quot;D&quot;100 ML, 솔루션의 10 ML &quot;C&quot;해결책 &quot;B&quot;10 ML 및 솔루션 10 ML &quot;&quot;. 세 initia비타민 리터 솔루션 (F, G, H)는 다음과 같이 준비가되어 있습니다 : <ul><li> 솔루션의 리터 증류수에 비오틴의 100 밀리그램이 들어있는 &quot;F&quot;; </li><li> ; 증류수에있는 티아민의 10g를 포함하는 솔루션 &#39;G&#39;의 리터 </li><li> 솔루션을 증류수에 cobalamin의 100 밀리그램을 포함하는 &quot;H&quot;의 리터. </li></ul> 비타민 (솔루션 &#39;I&#39;)의 최종 솔루션의 리터는 증류수로 세 초기 솔루션의 10 ML을 추가 얻어진다. algal 미디어 리터은 영양소 솔루션 미네랄 최종 해결책 (솔루션 &quot;E&quot;)와 해수의 비타민 최종 해결책 (솔루션 &quot;나는&quot;) 1 ML 1 ML 1 ML을 해소하여 준비가되어 있습니다. 이 후자의 인식 분석 품질 시약 또는 증류수 또는 드 이온화 물에 시판 제제를 분해하여 얻을 수 있습니다. 이상적인 바닷물을 만들기위한 소금 조화가 상업적으로 사용할 수 인스턴트 오션 섞어 예. 그것은 recommen입니다30 염도 (± 2) PSU 및 20의 온도 (± 1) ° C.로 해수를 사용을 ded

long-term lethal toxicity test with the crustacean artemia franciscana

우리의 연구 활동은 바닷물 / 소금기있는 물 및 침전물에 대한 특정 참조와 함께, 환경 품질 평가를위한 생물 학적 방법의 사용을 타겟팅합니다. 생물 학적 지표의 선택은 표준화된 절차의 예약 상황에, 가능성, 신뢰성있는 과학적 지식을 기반으로해야합니다. 이 문서에서 우리는 화학 물질 및 / 또는 해양 환경 매트릭스의 독성을 평가하기 위해 해양 갑각류 Artemia를 사용한 표준화된 프로토콜을 소개합니다. 과학자들은 소금물 새우 (Artemia)는 전 세계적 활용에 대한 표준 bioassay의 발달에 적합한 후보는 것을 제안합니다. 논문의 숫자는 소금물 새우 (Artemia)에서 다양한 화학 물질과 toxicants의 독성 효과에 공개되었습니다. 독성 연구를 위해이 갑각류의 주요한 이점은 건조 cysts의 전반적인 가용성이며 이들은 즉시 테스트에 사용될 수 있으며, 어려운 재배는 요구하지 않습니다 <suP&gt; 1,2. 낭종 기반 독성 assays이 저렴하고, 지속 가능한 간단하고 신뢰성 및 산업 모니터링 요건이나 규제 목적 3, 따라서 독성 검사의 일상적인 요구에 중요한 대답입니다. 제안된 방법은 엔드 포인트로서 사망률과 관련이 있습니다. 생존자의 숫자는 집계되었으며 사망자의 비율이 계산되었다. 그들이 관찰 4의 몇 초 동안 내부 또는 외부 움직임을 전시하지 않은 경우 애벌레는 죽은 것으로 간주되었다. 이 절차를 참조 물질 (나트륨 Dodecyl 황산) 표준 테스트를했습니다, 일부 결과는이 작품에보고됩니다. 이 문서는 프로토콜에 관련된 모든 단계를 보여주는 절차적 독성 테스트의 성능을 설명하는 동영상을 함께.

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

이 연구, 화학 물질, 산업 폐수 또는 하수 및 해수 갑각류에 액상 환경 시료에 의해 진력 장기 (14 일) 치명적인 독성을 결정하는 중요한 방법론 프로토콜의 개발 및 표준화에 관한<em&gt; Artemia franciscana</em&gt;.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

19.5K 조회수.  Institute for Environmental Protection and Research. 이 연구, 화학 물질, 산업 폐수 또는 하수 및 해수 갑각류에 액상 환경 시료에 의해 진력 장기 (14 일) 치명적인 독성을 결정하는 중요한 방법론 프로토콜의 개발 및 표준화에 관한 Artemia franciscana.

비디오: Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

광학 프로젝션 Tomography를 사용하여 인간 유방암 표본과 그 분석의 장기 문화

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

의 장기, 고해상도 공 촛점 시간 경과 영상<em&gt; Arabidopsis 떡잎</em발아시&gt; 표피

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

장기 성인 마우스 SSC 라인의 유도를위한 문화 Spermatogonial 줄기 및 전구 세포의 일련 농축 (SSCs)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

Amphipod 갑각류의 여덟 세포 배아에서 단일 셀의 절제<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

다층 제브라 피쉬의 장기 라이트 시트 현미경에 장착

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA, 장기 시간 경과 이미징을 위한 초파리 유충을 장착하는 방법

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

설치류의 머리에서 정의된 가속도를 생성하기 위한 수동 머리 움직임의 적용

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

실험실 조건에서 잎 절단 개미 (Atta)의 수집 및 장기 유지 관리

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...