저자는 mRNA의 추출 및 PCR과의 도움 폴 박사 알면서하는 씨와 제네 비브 선생을 인정하고자 및 지원에 대한 가넷 패스포트와 로드니 윌리엄스 기념 재단.

윤리 진술 : 아래에 설명 된 모든 절차는 시드니 동물 윤리위원회의 대학에 의해 승인되었다. 1. 동물 주 : 1 8~9개월 된 마우스 (C57 / BL6)는 동물 자원 센터 (퍼스, 호주)에서 얻었다. 마우스는 시드니 대학의 보쉬 설치류 시설에 수용되었다. <ol><li> 물과식이에 액세스 할 수있는 12/12 시간 빛 / 어둠주기에 표준 마우스 케이지 하우스 마우스. </li><li> 근적외선에 각 연령 그룹에서 나누기 마​​우스 (NIR) 처리, 또는 가짜 비교를 위해 (제어) 그룹을 처리 하였다. </li></ol> 2. 근적외선 (NIR) 조사 및 위장 치료 <ol><li> 처리 체제의 완료 전에 모발의 빠른 재성장을 막기 위해 가능한 한 가깝게 전기 면도기와 마우스 두경부에 털을 면도. 마우스의 무균 상태를 유지하기 위해, 70 % 에탄올을 사용하여면도 동물과 별도의 케이지에서 동물 사이의 F10 수의학 살균제 사이에 악기를 청소합니다. 동물 오버 처리되지 않도록 치료의 시작 3 일전 -이 2 마십시오. </li><li> 꼬리의 근위 끝을 잡고 마우스를 제지 및 false 결과로 이어질 수있는 스트레스를 최소화하기 위해이 한 손 또는 벤치 탑의 손바닥에서 휴식을 취할 수 있습니다. </li><li> LED NIR (670 ㎚) (발광 다이오드) 1 장치 잡고 - 2 ㎝ 떨어져 노출 (음모) 지역에서을 90 초 (그림 1A)의 장치에 전환합니다. 참고 : 90 초 노출의 결과로서 온도 변화를 물 100ml에 0.2 ° C &lt;로서 측정 하였다. </li><li> 동물의 가짜 치료 그룹에 대해 2.3하지만 장치가 꺼져 (그림 1B)를 떠나 - 반복 2.2 단계를 반복합니다. </li><li> 동물의 근적외선 차단 치료 그룹의 2.3하지만 알루미늄 호일로 장치를 커버 - 반복 2.2 단계를 반복합니다. </li><li> approximat 매일 2.5 - 반복 2.2 단계5 일 연속 전자 24 시간 간격. </li><li> 다섯째 날 후 처리에 모두 귀에서 전정 감각 상피 (3의 cristae 1 utricular의 황반)을 추출 (아래 3 항 참조). </li></ol> 3. 조직 추출 (20) <ol><li> (26)의 NaHCO3, 11 포도당, 250 글리세롤, 2.5의 KCl, 1.2의 NaH 2 PO 4, 1.2의 MgCl 2, 2.5 염화칼슘 2 : (mm 단위)로 구성된 글리세롤 기반 인공 뇌척수액 (ACSF) 300 ml의를 준비합니다. CaCl2를 첨가하기 전에, 7.4의 pH를 확립 칼슘 침전 (흐림)를 피하기 위해 carbogen (95 % O 2 및 5 % CO 2)와 용액을 기체. 얼음 슬러리가 형성되도록 45 분 동안 -80 ℃ 냉동고에서 용액을 냉각. </li><li> 표시된 나사 상단 모세관의 RNA 분리 용해 버퍼를 준비하는 것은 (뚜껑 터지는를 중지 할 때 동결 및 액체 질소에 해동 사이의 튜브 압력 변화)에 따라제조업체 또는 일반적인 실험실 관행의 지시에. 알루미늄 듀어 플라스크에 액체 질소를 준비합니다. 참고 : 액체 질소를 처리 할 때 보호 복 및 보호 eyeware을 사용합니다. 그 가스 형태의 볼륨이 약의 액체 형태의 700 배이며, 질식의 원인이 될 수 있습니다 액체 질소는 환기가 잘되는 방에서 사용되어 있는지 확인합니다. </li><li> 깊이 복강 내 주사를 통해 케타민 (400 ㎎ / ㎏)와 쥐를 마취시키다. 뒷다리 반사가 완전히 마우스가 완전히 마취되어 있는지 표시로 가라 앉을 수 있습니다. </li><li> 날카로운 스테인레스 스틸 가위로 마우스를 목을 벨과 면도날을 사용하여 두개골의 시상 피부를 따라 절개를 (# 22 반올림). 이 시점에서과를 통해 3.5 단계 - 3.9, 조직을 통해 얼음처럼 차가운 ACSF의 일반 응용 프로그램에서 가능한 한 멋진 두개골 금고, 뇌 및 기본 전정 장치를 유지합니다. </li><li> 에 작은을 표준 패턴 가위의 뾰족한 팔을 사용하여리터 람다의 두개골 절개 및 시상 봉합을 따라 잘라. </li><li> 부드럽게 뇌를 끌어없이 뼈의 하부 표면에 가능한 한 가깝게 블레이드를 유지 정수리 뼈 아래에 얕은 rongeurs의 한 팔을 밀어 넣습니다. 안전하고 뇌가 노출 될 때까지 후방 측면 정수리 뼈와 후두골을 당깁니다. </li><li> 작은 스테인레스 스틸 주걱을 사용하고 vestibulocochlear 신경 (CN VIII)을 노출 멀리 전방 및 중간 두개골 포사에서 뇌를 들어 올립니다. 직접 전정 모발 세포에 분포 일차 구 심성 축색에 불필요한 장력 않도록 신경을 횡단면. </li><li> CN VIII의 절개 후 토토의 뇌를 제거합니다. </li><li> 달팽이관 중간 두개골 포사의 주변 전정 기관을 포함하는 뼈 미로를 관찰합니다. 각각의 뼈 미로 옆에 두 개의 작은 절개를 확인하고 앞쪽에 반원형 운하를 누른 후 잡아 당겨 전체 구조를 절제동맹. </li><li> 즉시 지속적으로 carbogen와 관류 동안 (3.1 단계에 설명 된대로) 얼음처럼 차가운 ACSF 솔루션을 포함하는 해부 접시에 절제된 미로를 체험. </li><li> 실체 현미경에서 달팽이관으로 미로를 누른 상태에서 집게로 접시의 바닥에 고정합니다. <ol><li> 전방 반원의 운하 (SSC) 팽대부 위의 뼈에 작은 구멍을 긁어 직선 미세 집게를 사용합니다. </li><li> 부드럽게 즉시 뼈 아래에 도달 바깥쪽으로 멀리 팽대부에서 쓸어 넘겨이 개방을 확대. 구멍에 집게를 밀어 아래 깨지기 쉬운 막성 미로를 손상하지 않도록 여기에주의하십시오. 난형 낭, 전방 및 측면 ampullae 모든 노출 될 때까지 이런 식으로 계속합니다. 가능하면 또한 후방 팽대부를 제거합니다. </li></ol></li><li> 미세 집게를 사용하여 부드럽게 난형 낭을 들어 올려가 완전히 분리 될 때까지 뼈 미로에서 멀리 ampullae. 가능하면 그들을 붙잡아관련 반원의 운하에 의해 감각 상피의 손상을 방지합니다. 어떤 경우에는, 반원형 멤브레인 덕트의 기단 부분은 뼈 ampullae를 해제 홍채 가위로 절단 할 필요가있다. </li><li> 안전하게 단계 3.2에서 이전에 제조 된 용해 버퍼에 포셉의 팁과 장소 사이의 전정 기관을 파악. 부드럽게 전정 기관이 집게에서 분리 한 보장하기 위해 버퍼에 주위에 집게를 소용돌이 친다. 이 실체 현미경 집게를 가져 와서 경우가 확인합니다. <ol><li> 미세 소관 뚜껑에 나사 즉시 액체 질소에 샘플을 동결. </li></ol></li></ol> 4. RNA 추출 및 RT-PCR <ol><li> 제조업체의 지침 또는 선호하는 실험실 프로토콜에 따라 메신저 RNA (mRNA의) 추출의 표준 방법을 따르십시오. 참고 : mRNA의 작은 수율을 분리 할 수​​ 있도록 캐리어의 RNA를 활용 한 상용 키트이 사용되었다프로토콜입니다. 낮은 용출 볼륨은 mRNA의 최종 농도를 증가시키기 위해 사용될 수있다. 참고 : 음 &quot;아니오 효소&quot;컨트롤 (NECs)와 &quot;아니오 DNA 템플릿&quot;컨트롤 (NTCS)도 관찰 효과의 유효성을 확인하기 위해 완료해야합니다. </li><li> 표적 유전자 21-23 보완 DNA (cDNA를) 증폭에 mRNA를 역전사의 표준 방법을 적용합니다. </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+induces+oxidative+stress+and+decreases+endocytosis+and+monolayer+permissiveness+in+cultured+kidney+epithelial+cells+without+affecting+viability.">Myoglobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability.</a> Kidney and Blood Pressure Research. 31, 16-28 (2008).</li><li>Saee-Rad, S., 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=Analysis+of+superoxide+dismutase+1,+dual-specificity+phosphatase+1,+and+transforming+growth+factor,+beta+1+genes+expression+in+keratoconic+and+non-keratoconic+corneas.">Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas.</a> Molecular vision. 19, 2501-2507 (2013).</li><li>Albert, E. S., 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=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> Journal of neurophysiology. 107, 3227-3234 (2012).</li><li>Moro, C., 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=Photobiomodulation+inside+the+brain:+a+novel+method+of+applying+near-infrared+light+intracranially+and+its+impact+on+dopaminergic+cell+survival+in+MPTP-treated+mice.">Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.</a> Journal of neuroscience. 120, 670-683 (2014).</li><li>Moreno, L. 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=Infrared+neural+stimulation:+beam+path+in+the+guinea+pig+cochlea.">Infrared neural stimulation: beam path in the guinea pig cochlea.</a> Hearing research. 282, 289-302 (2011).</li><li>Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+red+thread+as+a+guide+in+the+vestibular+labyrinth.">A red thread as a guide in the vestibular labyrinth.</a> The Journal of physiology. 589, 1241-1241 (2011).</li><li>Chakrabarti, S., 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=Mitochondrial+Dysfunction+during+Brain+Aging:+Role+of+Oxidative+Stress+and+Modulation+by+Antioxidant+Supplementation.">Mitochondrial Dysfunction during Brain Aging: Role of Oxidative Stress and Modulation by Antioxidant Supplementation.</a> Aging and disease. 2, 242-256 (2011).</li><li>Petrosillo, G., De Benedictis, V., Ruggiero, F. M., Paradies, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decline+in+cytochrome+c+oxidase+activity+in+rat-brain+mitochondria+with+aging.+Role+of+peroxidized+cardiolipin+and+beneficial+effect+of+melatonin.">Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin.</a> Journal of bioenergetics and biomembranes. 45, 431-440 (2013).</li><li>Zhu, H., Sun, A., Zou, Y., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+metabolic+adaptation+promotes+mesenchymal+stem+cell+therapy+for+ischemia:+a+hypoxia-induced+and+glycogen-based+energy+prestorage+strategy.">Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia: a hypoxia-induced and glycogen-based energy prestorage strategy.</a> Arteriosclerosis, thrombosis, and vascular biology. 34, 870-876 (2014).</li></ol>

저자는 그들이 더 경쟁 재정적 이해가없는 선언합니다.

여기에 설명 된 대표 결과 근적외선 간략한 두개 납품 (오일을 90 초 / 일) 가성 처리 한 마우스와 비교했을 때 이전 마우스에서 항산화 제의 발현 수준을 인상하기에 충분한 것으로 나타났다. NIR에 의해 방출되는 열을 우리의 측정 장치가 &lt;0.2 ° C 초 90이었다 LED와 같은을 일으킬 가능성이 - 방출되는 열이 미토콘드리아 및 / 또는 신경 활성화의 소스를 나타낼 수 있지만, 쥐의 전정에 대한보고는 <...

밸런스 성능을 떨어지는 이후의 폭포는 일반적인, 자연 노화 하나의 불행하게도 종종 정의하는 기능을 제공합니다. 이 감소의 영향은 신체적, 사회적인, 그리고 크게 노인을위한 삶의 질을 줄일 수 있습니다. 응답에서, 물리적 치료와 재활 폭포에 연구의 초점이되어왔다하지만 반복 폭포의 유병률 일관된 감소와 연관되지 않았다. 동시에, 작업은 말초 또는 중추 전정 시스템 (균형을 유지할 책임이있는 시스템) 부족, 및 이러한 시스템을 대상으로 잠재적 인 치료 전략의 변경 및 한정 불균형의 근본 원인을 조사. 연령 관련 황반 변성 2-4, 알츠하이머 병 모델 5-8, 파킨슨 병 9-12 포함하여 연령 관련 퇴행성 신경 질환에 대한 최근 연구는시의 신경 보호 효과를 보여 주었다근적외선 (NIR) 빛의 mple 비 침습적 응용 프로그램입니다. 또한, 전정 시스템에서, NIR 시험관 13 전정 일차 구 심성 뉴런의 활성을 증가시키는 데 사용되어왔다. 근적외선의 메카니즘은 잘 이해되지 않지만, 근적외선을 사용하여 대부분의 연구는 근적외선 세포 대사를 촉진하는 미토콘드리아 복잡한 IV (사이토 크롬 c 산화 효소) 14-17을 자극한다고 제안했다. 전정 감각 상피에서 I 형 머리 세포의 표피 하 판은 미토콘드리아 (18)의 조밀과 같은 치료 근적외선 치료를위한 행동의 사이트를 나타낼 수있다. 여기서, transcranially의 간단한, 비 침습성 치료 체제는 세포 대사를 측정하는데 사용될 수있다 (그리고 암시 미토콘드리아 기능에 의해) 마우스 전정 감각 상피 설명 NIR을 적용했다. 또한 논의 전정 감각 상피 제제이며 근적외선 ubiquito의 발현을 증가 시킨다는 것을 나타낸다우리는 항산화 감각 상피 (슈퍼 옥사이드 디스 뮤 타제 1) - 이전에 와우 헤어 세포 생존 (19)에 대한 중요한 것으로 나타났다.

<table border="0" cellpadding="0" cellspacing="0" width="603">
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
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			<td height="42" style="height:42px;">Quantum WARP 10</td>
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			<td height="63" style="height:63px;">Screw top microtubules</td>
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			<td></td>
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			<td height="63" style="height:63px;width:216px;">Ketamine</td>
			<td style="width:101px;">Parnell, Alexandria Australia</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Standard Pattern Scissors</td>
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			<td height="21" style="height:21px;width:216px;">Friedman-Pearson Rongeurs</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">16221-14</td>
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			<td height="42" style="height:42px;width:216px;">Stereo microscope</td>
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			<td height="21" style="height:21px;width:216px;">Dumont #5 SF Forceps</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">11252-00</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
			<td style="width:101px;">Bioline</td>
			<td style="width:119px;">BIO-52075</td>
			<td></td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

연령이 증가함에 따라 밸런스 기능에 감소를 감쇠위한 전략 밸런스 작업과 운동을 포함하여 물리 치료에 주로 초점을 맞추고 있습니다. 그러나 이러한 접근 방법은 균형 하락의 근본 원인을 해결하지 않습니다. 마우스를 사용하여, 전정 감각 상피 세포의 대사에 근적 외광의 영향 (NIR)을 평가 하였다. 수집 된 데이터는이 간단하고 안전 개입이 자연 노화의 해로운 영향으로부터이 취약한 세포를 보호 할 수 있음을 보여줍니다. 의 mRNA는 격리 된 주변 전정 감각 상피 (크리스타의 ampullaris 및 utricular의 황반)에서 추출 이후 cDNA 라이브러리로 전사했다. 이 라이브러리는 다음 유비쿼터스 항산화 제 (SOD-1)의 발현을 조사해서되었다. 항산화 유전자 발현 후 세포 대사를 정량화하는 데 사용되었다. 젊은에서 NIR의 두개 배달 (4 주) 이상 사용 - 5 D (8 9 개월) 마우스 및 간단한 치료 정권 (90 초 / 일AYS),이 작품은 전정 감각 상피 세포에서 미토콘드리아의 기능을 개선하기에 충분할 수있다 혼자 NIR을 제안한다. 전정 모발 세포 기능을 개선하기위한 치료의 사용 가능한 저렴한, 비 침습적 방법은 없기 때문에, 외부 근적외선의 애플리케이션은 전정 감각 상피 기계가 세포 대사에 노화의 영향을 상쇄 할 가능성 전략을 제공한다.

젊은에서 NIR 처리의 영향 (4 주) 이상 비교하기 - 쥐 (8 9 개월) 우리는 (N = 20) (N = 16) 젊은에서 항산화 슈퍼 옥사이드 디스 뮤 타제 1 (SOD-1)의 발현을 측정 이상 가짜 처리, NIR 처리, 또는 근적외선 차단했다 쥐.이 그림은 젊은 가짜 처리 된 동물에 비해 NIR 처리 어린 동물에서 2 개 이상의 배의 β - 굴지 정규화 SOD-1의 발현이 크게 증가를 보여줍니다 (P &lt;0.01) 및 젊은 근적외선 차단 동물 (P...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

미토콘드리아 기능 장애는 세포 노화의 특징이다. 이 논문은 노화 마우스 전정 감각 상피의 미토콘드리아 기능을 개선하기 위해 비 침습적 근적외선 (NIR) 치료를 사용합니다.

근적외선 (NIR) 빛은 마우스 전정 감각 상피 세포에서 미토콘드리아 기능의 마커의 발현을 증가

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

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Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K 조회수.  University of Sydney.  미토콘드리아 기능 장애는 세포 노화의 특징이다. 이 논문은 노화 마우스 전정 감각 상피의 미토콘드리아 기능을 개선하기 위해 비 침습적 근적외선 (NIR) 치료를 사용합니다. 

비디오: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

가까운 적외선 분광학과 함께 아기, 어린이 및 성인의인지 기능을 탐색

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

연구

생물학

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

인간 iPS 세포의 마이크로 RNA 발현 프로파일, IP의 유래 망막 색소 상피 세포, 태아의 망막 색소 상피

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

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

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

에서 미토콘드리아의 구조와 기능을 연구<em&gt; 초파리</em&gt; 난소

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

마우스 망막 안료 상피에서 1 차 셀 문화

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...