우리는 촬영에 그들의 도움을, 조지 자비스, 마사 복, 그리고 베티 Hecox - 레아, 해양 생물 연구소에 감사하고 있습니다.

미디어의 1. 준비 참고 : 사용에게 염분 16.5 PPT (PSU)의 반 희석 Brujewicz 인공 해수를. 다른 인공 seawaters도 자주 문화 Brachionus의 로티퍼 (25), (26)에 사용된다. <ol><li> 454 mM의 염화나트륨을 추가, 26 밀리미터의 MgCl 2, 27 mM의 황산, 10 밀리미터의 KCl, 증류수 10 mM의 CaCl2를 4.5 L은 (최종 부피 5 L 될 것입니다). 또한, 탈 희석 물 대신에 증류수를 사용합니다. 다른 모든 염을 용해 후 CaCl2를 추가합니다. </li><li> 0.48 M의 NaHCO3 원액 (200 배 농도)를 준비합니다. 상기 용액에 25 ML을 추가합니다. NaHCO3을 최종 농도가 2.4 mm이다. </li><li> 0.4 M NaBr은 원액 (500 배 농도)를 준비합니다. 상기 용액에 10 ㎖를 추가합니다. NaBr은 최종 농도가 0.8 mm이다. 증류수로 5 L까지합니다. </li><li> 0.45 ㎛ 멤브레인 필터 솔루션 필터.사용하기 전에 그것을 멸균 수로 2 회 희석 (v / v)로. 참고 :이 원액으로 Brujewicz 인공 해수의 2 배 농도를 할 수있다. </li></ol> 2. 일반 문화 조건 <ol><li> 문화 실험실 제기 또는 20 및 30 °의 C 사이의 멸균 100 ㎖ 비이커에 야생 캡처 로티퍼. 높은 온도는 더 짧은 수명 가속 재생을 초래한다. 실험의 편의를 위해 25 ° C를 사용합니다. 로티퍼 밀도 또는 인공 해수의 부피는 여기에 큰 문제가되지 않습니다. </li><li> 인공 해수 (11) 문화식이 미세 조류 종. 스넬 등의 알을 참조하십시오. (2014) 자세한 내용은 11. 일반적으로, Tetraselmis의 tetrathele 사용 (~ 2 × 10 5 세포 / ㎖ 27,28), T. suecica (~ 6 × 10 5 세포 / ㎖ 25,29), 및 Nannochloropsis의 oculata (~ 7 × 10 6 세포 / ㎖ 30, 31). 다이어트 조류 때문에 (종, 문화 CONDIT이온, 생화학 적 조성물은) 상당히 모든 실험군에서 microalga 같은 많이 사용, Brachionus의 로티퍼의 생존 기간에 영향을 미친다. </li><li> 먹이 (예를 들어, 2 일마다 먹이를 매주 접종) 가끔 미디어를 변경하여 배치 문화에 로티퍼의 재고 인구를 유지합니다. 로티퍼가 최적의 상태에있을 때 많은 신생아 2-3 계란 베어링 성인 관찰 할 수있다. 참고 : 실험 로티퍼를 얻기 위해 또 다른 방법은 휴식 달걀을 부화하는 것입니다. 휴식 계란에서 부화 로티퍼 잘 동기화 된 것으로 간주, 그들의 수명은 amictic 계란 (11) 부화 로티퍼의 그것과 크게 다르지 않다된다. 그러나, 휴식 계란에서 로티퍼 이전 amictic 계란에서보다 재생을 시작합니다. 따라서,주의는 생식 특성의 측정이 필요하다. </li></ol> 사전 문화에 의해 로티퍼 3. 동기화 <ol><li> 보안 목표 명세서에서 하나의 로티퍼를 선택옥토퍼스의 인구와 문화로 실험에 사용되는 하위 집단을 설정 2.1-2.3에 설명. 2 주 동안 일반적으로 문화. </li><li> 하위 집단에서 계란 베어링 로티퍼를 수집 (실험에 사용되는 개인의 이중 번호를 수집). 단일 코호트 같은 배양을 (농도 : ~ 50 개인 / ㎖) 광고 무제한 공급하에 신선한 배지에서 2.1-2.3에서 설명한 바와 같이, 6 웰 배양 플레이트이다. 조절 된 매체가 Brachionus의 로티퍼 (32, 33)의 생식 생리에 영향을주기 때문에 인구 밀도를 제어하는 것이 중요합니다. </li><li> 새로 준비 문화 매체에 성인의 첫번째 계란에서 부화 신생아를 전송합니다. 2 ~ 3 세대에 걸쳐이 절차를 반복합니다. </li><li> 생존 시간을 측정하기위한 소정의 시간주기 (예를 들어, &lt;3 시간)에서 빗금 신생아를 사용한다. , 색인 전압 여러 가지를 사용하여 재현성을 확인 매우 희한하지만, 하나의 개인을 선택할 가능성 편견을 방지하기 위해늘어진 하위 집단. 참고 : 로티퍼는 일반적으로이 조건으로 만 무성 생식으로, 어떤 남성이 실험을 통해 존재하지 않는 확인합니다. 남성은 신생아보다 작은 일반적으로 빠른 여성보다 이동합니다. Mictic 여성은 특정 조건 (29)에서 amictic 여성 다른 수명을 가지고있다. </li></ol> 생존 시간 4. 측정 <ol><li> 플라스틱 접시에 배치 신생아 (인공 해수의 각 웰 포함 1 mL를 일반적으로 24 또는 48 웰 플레이트). </li><li> 24 시간 간격으로, 만약 높은 온도 (위 30 ° C 이상)에서 새로 제조 된 배양 배지 또는 12 시간 간격으로 로티퍼를 전송합니다. 자손의 수를 기록하고 각 개인은 죽은이나 살아 있는지 여부. 코로나의 섬모 운동이 완전히 정지했을 때 죽은로 로티퍼를 기록합니다. 참고 피퍼 종종 웰의 측벽에 부착. 물을 부드러운 피펫 그들을 찾는 데 도움이됩니다. 로티퍼를 찾을 또는 아칸소되지 않은 경우전자는 실수로 &quot;검열​​&quot;라고하지 &quot;죽은&quot;로, 그들을 기록, 피펫에 의해 손상. </li><li> 실험 로티퍼 적극적으로 재생 될 때 신생아를 제거합니다. 신생아 빠르게 성장하고 실험 피퍼 구별 때때로 어렵다. </li></ol> 5. 데이터 분석 <ol><li> 카플란 - 마이어 생존 곡선 (도 1 및 2)를 X 축, Y 축 및 시간을 누적 생존율을 플로팅하여 만들기. 이 생존 데이터의 일반적인 표현이다. 생존 시간 (34)의 통계 비교 (또한 벽난로 - 콕스 테스트라고도 함) 비모수 로그 순위 테스트를 사용합니다. 로그 순위 테스트는 또한 JMP와 R. 같은 다른 표준 통계 패키지에 포함되어 있습니다 주 : 정규 분포는 일반적으로 생존 데이터 (35)에 의해 충족되지 않기 때문에 학생의 t- 테스트 또는 파라 메트릭 다중 비교 다음 분산 (ANOVA) 분석을 사용하지 마십시오. 또한, 이러한 운전 방식DS는 계정에 검열 개인을하지 않습니다. 더 검열 데이터가없는 경우 맨 - 휘트니 U 시험을 이용할 수있다. </li></ol>

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Sci. 68 (4), 349-358 (2013).</li><li>Kaneko, G., Kinoshita, S., Yoshinaga, T., Tsukamoto, K., Watabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+expression+patterns+of+stress+protein+genes+during+population+growth+of+the+rotifer+Brachionus+plicatilis.">Changes in expression patterns of stress protein genes during population growth of the rotifer Brachionus plicatilis.</a> Fish. Sci. 68 (6), 1317-1323 (2002).</li><li>Kim, H. J., Sawada, C., Hagiwara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+and+reproduction+of+the+rotifer+Brachionus+plicatilis+species+complex+under+different+light+wavelengths+and+intensities.">Behavior and reproduction of the rotifer Brachionus plicatilis species complex under different light wavelengths and intensities.</a> Int. Rev. Hydrobiol. 99 (1-2), 151-156 (2014).</li><li>Yoshinaga, T., Hagiwara, A., Tsukamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conditioned+media+on+the+asexual+reproduction+of+the+monogonont+rotifer+Brachionus+plicatilis+O.+F.+Müller.">Effect of conditioned media on the asexual reproduction of the monogonont rotifer Brachionus plicatilis O. F. 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The authors have nothing to disclose.

현재의 프로토콜 Brachionus 피퍼의 생존 시간을 측정하는 방법을 설명한다. 중요한 단계는 여러 세대에 걸쳐 로티퍼 상태의 동기화이다. 실험 피퍼 잘 동기화되면 여러 선행 연구 18,24,37,38에보고 된 일반적인 유형 I 생존 곡선은 거의 조기 사망이 관찰된다. 생존 시간의 표준 편차 따라서 높은 통계 전력의 결과로 가난하게 동기화 로티퍼에 비해 작아진다. 동기화는 또한 생존 시간 측정의 재현성을 향상 할 것으로 예상된다 - 엄마 최적 조건에서 배양되므로, 현재 프로토콜 모체 세대의 가능한 해로운 효과를 상쇄. 조기 사망률이 여전히 조심 동기화 후 관찰되는 경우, 새로 준비 문화 미디어 사용을 고려, 즉 먹이 조류, 또는 새로 설립 된 실험 집단의 다른 많은 (,) 프로토콜 3.1에서 시작합니다. 이 프로토콜의 제한은 잘 동기화 로티퍼 잠재적 통해 문자를 구분한다는 것입니다. 예를 들어, 수명을 연장 화학 물질의 심사에 따라,이 프로토콜에 의해 상영 일부 화학 물질은 제대로 동기화 로티퍼의 수명에 심각한 영향을 감지하지 못할 수 있습니다 (예를 들어, 야생 및 배치 배양 인구에서 개인). 따라서, 이러한 실험 결과는 신중하게 해석되어야한다. 자손의 생존 기간에 산모의 나이의 효과는 과일 파리 초파리 melanogaster의 및 Caenorhabditis 엘레은 39, 40 엘레 간스 선충을 포함한 다른 무척추 동물 모델에서보고되었다. 더 시간 소모적이 장수 모델이지만 이들 동물이 생존 시간의 측정 실험에서의 변화를 감소시키기 위해 여러 세대에 걸쳐 동기화 절차에 유용하다.

로티퍼는 문 Rotifera 1을 구성하는 미세한 국제 동물 플랑크톤 (&lt;1mm)입니다. 이들은 약 1000 체세포과 특성 차륜 형 섬모 장치 이루어지는 간단한 신체 운동 계획 및 공급을 위해 사용되는 코로나라고있다. 대부분의 로티퍼는 약 1,600과 500 종 각각 2를 포함하는 클래스 단 소강 또는 질 형목에 속한다. bdelloid 로티퍼가 의무 처녀 생식 (3)에 의해 재생하는 동안 단 소강 로티퍼는 일반적으로 모두 성적과 무성 생식 단계 (순환 처녀 생식)가 있습니다. 이 실험에서 높은 재현성을 보장 전적으로 동일한 로티퍼 개인을 얻는 것이 가능하다. 또한, 그들은 짧은 수명, 배양 용이성, 게놈과 transcriptomic 4-7 시퀀스 데이터의 가용성, 및로부터 떨어진 독특한 계통 위치로서 모델 생물 여러 다른 장점을 가지고rthropods 및 선충 8. 로티퍼 따라서 생태 학적, 독성의 무척추 동물 모델을 약속하고, 연구 9-12 노화된다. 환경 스트레스 또는 화학 물질에 노출 하에서 생존 시간이 13-19 연구 분야에서 자주 측정 된 파라미터이다. 이 어머니의 환경 조건에 민감하기 때문에 피퍼의 생존 시간을 측정하는 경우에는주의가 필요하다. 즉, 단 소강 로티퍼 Brachionus의 manjavacas에 세 어머니에서 여성 자손은 젊은 산모에서보다 짧은 수명을 가지고, 그러나 산모의 칼로리 제한 (CR)은 부분적으로 고급 산모의 나이 (20)의 해로운 효과를 상쇄. B.에서 plicatilis, 산모 CR은 자손의 수명, 기아에서 오래 생존 기간 및 항산화 효소 (21, 22)의 강화 된 표현과 관련된 높은 산화 스트레스 저항을 제공한다. 임산부 연령 효과또한 bdelloid 로티퍼 (23)에서 관찰되었다. 따라서, 실험 로티퍼의 조건을주의 깊게 생존 시간을 측정하기 전에 몇 세대에 걸쳐 동기화되어야합니다. 여기에서 우리는 여러 세대에 걸쳐 배양 조건의 동기화 다음 Brachionus의 로티퍼의 생존 시간의 측정을위한 프로토콜을 제공합니다. 간헐적 단식 (IF), 피퍼 주기적으로 공급되는 CR의 변형은 22, 24에 의한 장수 IF의 잘 알려진 효과 동기화의 효과를 나타 내기 위해 적용 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">190-13921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Magnesium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">136-03995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Magnesium sulfate</td>
			<td style="width:135px;">Wako</td>
			<td>131-00427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Potassium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">168-22111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Calcium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">035-00455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium bicarbonate</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">199-05985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium bromide</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">190-01515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Membrane filter (0.45 &#181;m pore size)</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:119px;">HAWP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Culture plate, 6-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D01</td>
			<td>Flat bottom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Culture plate, 48-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D07</td>
			<td>Flat bottom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Tetraselmis, Living</td>
			<td style="width:135px;">Carolina Biological Supply Company</td>
			<td style="width:119px;">152610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">PRISM 6</td>
			<td style="width:135px;">GraphPad Software</td>
			<td style="width:119px;">Version 6.0d</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of survival time in brachionus rotifers: synchronization of maternal conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

그림 1은 제대로 동기화 인구 (두 개의 복제 만점)의 대표적인 생존 곡선을 보여줍니다. 이 실험에서, 로티퍼이었다 중 공급 일상 [광고 무제한 (AL) 그룹] 또는 (그룹 IF) 매일. 평균 생존 기간은 각각 그룹화 AL 및 IF (13) 18 일이었다. 이를 잘 IF는 로티퍼의 수명을 연장하는 것이 알려져 있지만,이 실험은 AL 및 그룹 IF 수명 사이의 유의 한 차이를 검출하지 못했다. 경험적 조기 사망률과 생존율의 점진적인 감소가 불충분 동기화 결과는이 실험에서 관찰했다. 하위 집단에 대한 부적절한 처리 또는 낮은 수질로 인한 로티퍼 손상과 유사한 결과를 얻을하는 경향이있다. 로티퍼 조건이 최적 잘 동기화되면, 조기 사망은 거의 관찰되지 및 accordin된다의 Gly 로티퍼 실험 (그림 2)의 나중 단계에서 동기화 된 방식으로 죽는 경향이있다. 평균 생존 기간은 각각 그룹화 AL 및 IF (13) 20 일이었다. 적은 수의 동물이도 1의 실험보다 사용되었지만, 이들 그룹 간의 수명의 차이는 통계 학적으로 유의 하였다. 이것은 이전에 22 게시 된 개 이상 실험의 대표적인 결과입니다. <img alt="그림 1" src="/files/ftp_upload/54126/54126fig1.jpg" /> 그림 1 :.은 IF 그룹이 매일 공급 된 반면, 간헐적 단식 (IF)를 실시 제대로 동기화 개인에 대한 카플란 - 마이어 곡선은 AL 그룹은 실험을하는 동안 광고 무제한을 공급 하였다. N은 AL에 대해 11, N = 12 및 그룹 = 경우 각각 (N은 실험에 사용 된 개개의 개수를 나타낸다). 실험을 하였다 perfor의대 25 ° C에서. 로그 순위 테스트 (P = 0.1207)를 사용했을 때 수명에 유의 한 차이가 발견되지 않았다. 그러나,이 데이터는 시험 36 강력한 것으로 알려져 있지만 로그 - 랭크 시험을 두 횡단 생존 곡선을 비교하기 위해 사용되지 않아야하기 때문에 해석하는 것이 곤란하다. 확립 된 방법은 현재 검열 데이터와 생존 곡선을 교차 할 수 없습니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54126/54126fig2.jpg" /> 그림 2 :. IF를 실시 동기화 개인 카플란 - 마이어 곡선은 예비 배양에 의해 얻어진 로티퍼 코호트는, (매일 먹이) 같은 IF 일정을 하였다. N = 6, AL에 대한 N = 8 및 그룹 IF, 각각. 실험은 25 ℃에서 수행 하였다. 기록-순위 테스트, P = 0.0057. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

생존 시간의 측정에<em&gt; Brachionus</em&gt; 로티퍼 : 산모 조건의 동기화

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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Nanyang Technological University

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Biology

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

8.4K 조회수.  The University of Tokyo. Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

비디오: Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

연구

생물학

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

세포 생존에 박테리아와 그 영향의 동결 건조 공법

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

단일 및 다세포 상황에서 Invadopodia로 인한 세포 외 기질 Proteolysis의 양적 측정

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

의 동기화<em&gt; Caulobacter Crescentus</em&gt; 세균 세포주기의 조사에 대한

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

동기화를 필요로하지 않고 유동 세포 계측법을 사용하여 세포주기 진행의 시간적 추적

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...