이 작품은 MD에 NIH 부여 NS042197에 의해 지원되었다. AJK는 보조금 T32GM008719와 F30NS068006 지원했다.

1. Microinjection 니들스 생산 <ol><li> 사전 조립 microinjection의 바늘은 상업적으로 사용할 수 있습니다 (예. Eppendorf에서 Femtotips)와 하나 microinjections 많은 수의를 수행하지 않은 경우 유용합니다. 그러나, microinjecting위한 장기적인 기능을 구축하고자하는 사람들을 위해, 대안은 얇은 벽 borosilicate 유리 모세관과 상업 바늘 풀러를 사용하여 실험실에서 microinjection의 바늘을 생산하는 것입니다. 이것은 또한 다른 세포 유형에 유용할 수 있습니다 다양한하는 바늘의 모양을 수 있습니다. </li><li> Narishige PC - 10 Microinjection 니들 풀러으로, 네 명 모두 무게를 첨부 58.0에서 상대 열을 설정 (제 2 히터)로 한 단계 당기는 프로그램 (1 단계 설정)을 사용합니다. 두 결과 바늘이 비슷한 길이 수 있도록 각각의 모세관의 중심에서 가열 요소를 배치해야합니다. </li><li> 여러 모세관 (이자 각각의 단백질에 대한 모세 혈관 약 2) 당기세요. </li><li> 용기에 저장 바늘 바늘 팁을 손상하지 않도록하는 동안. 이러한 거품 또는 블루 - 테크와 같은 자료는 microinjection의 바늘을 가질 용기에 사용할 수 있습니다. </li></ol> 2. 사출을위한 단백질 혼합물의 준비 <ol><li> 7.4의 산도 1 M 염화칼륨 및 0.1 M의 칼륨 인산 (KPI) 버퍼 (K 2 HPO 4, KH 2 PO 4 몰 혼합)이있는 10X microinjection 버퍼를 준비합니다. 이 버퍼는 상온에서 장기간 저장할 수 있습니다. </li><li> 주사를 시각화하기 위해, rhodamine - dextran 같은 형광 염료가 추가되어야합니다. 물에 the10x microinjection 버퍼를 희석하여 20-40 MG / ML rhodamine dextran를 포함하는 배 microinjection 버퍼 솔루션을 만들기 위해 rhodamine - dextran 가루를 해산. </li><li> 4 어둠이 솔루션을 저장 ° C. 그것은 최대 100 개별 단백질 솔루션 준비에 대한 충분해야합니다. 플루오레신 isothiocyanate - dextran 같은 염료는 대체할 수 있습니다. </li><li> 20 MG / ML의 농도로 물에 정화 시토크롬 C를 용해하여 시토크롬 C 주식을 준비합니다. -80에서 보관 시토크롬 C ° C 장기 저장 및 소형 (~ 10 μL) aliquots에서 시토크롬 C를 저장하여 냉동 해동주기를 피하기 위해. </li><li> 1X 버퍼 (100 MM KCl, 10 MM KP 10 MG / ML 시토크롬 C의 최종 농도 3 μL 물 5 μL 시토크롬 C와 rhodamine - dextran을 포함하는 2 μL 배 microinjection 버퍼를 결합하여 사출을위한 10 μL 단백질 혼합물을 준비 I, 4-8 MG / ML rhodamine dextran). </li></ol> 3. 시토크롬 C의 세포질 Microinjection <ol><li> 직전 microinjection에, 4 10 분 16,000그램에서 시토크롬 C와 microinjection 버퍼의 혼합물을 원심 ° C는 microinjection의 바늘을 방해할 수있는 미립자 물질을 분리. </li><li> 원심 분리하는 동안, 공기 압력이 건설 수 있도록 microinjector 켭니다. </li><li> 현미경 단계의 중앙에 세포가 한 접시를 놓고 세포에 초점을 설정합니다. 세포 배양기 외부 유지되는 시간을 최소화하기 위해, 세포는 일반적으로 30 분 이내에 인큐베이터에 반환됩니다. </li><li> 모세관의 뭉툭한 끝에 단백질 혼합물의 상단 표면에서 피펫 0.5-1 μL. 튜브의 바닥에 centrifuged되었습니다 어떠한 입자를 피펫하지 않도록주의한다. 분 이내에 단백질 혼합물은 모세관 작용을 통해 바늘 끝에 배포됩니다. </li><li> micromanipulator과 위치의 팁 약 45 ° 각도에서 현미경으로 전송된 빛을 통과되도록 바늘의 모세관 홀더에 단단히 바늘을 연결합니다. </li><li> 그것이보기의 필드의 중심에 바로 위치해 있도록 바늘 끝의 위치를​​ 조정합니다. 센터 바늘을하려면, 현미경의 접안 렌즈를 통해 보면서 바늘을 이동 micromanipulator를 사용합니다. 바늘 그림자가 표시되어야합니다. 그 그림자에만 바늘 끝이 세포 위에 중심을 나타냅니다보기의 필드의 절반에서 볼되도록 바늘을 조정합니다. </li><li> 연속 흐름 모드로 microinjector를 설정하고 20 작업 압력 설정 - 100 고전력 증폭기 있습니다. 각각의 바늘은 다른 작업 압력을 요구할 수 있으며 작동 압력 가능성이 지속적으로 흐름을 유지하기 위해 microinjection 절차 중에 조정이 필요합니다. </li><li> 그냥 셀 위에 위치로 거친 노브를 사용하여 바늘을 낮춥니다. 이 작업을 수행하려면 셀 위에 위치로 현미경의 초점 비행기를 올립니다. 바늘 끝 초점 때까지 다음 굵은 손잡이를 사용하여 세포를 향해 바늘을 낮추십시오. </li><li> 다시 중심 바늘보기 분야 내에서 팁 및 현미경의 목적을 변경하여 배율을 향상시킬 수 있습니다. </li><li> 천천히 잘 속이는 사람 노프 unti를 사용하여 바늘을 낮출리터 바늘은 약간 세포의 초점 평면 위에있다. </li><li> rhodamine의 붉은 형광을보고하여 단백질 혼합물의 흐름을 확인합니다. 단백질 혼합물은 얇고, 지속적인 스트림으로 바늘을 종료해야합니다. 바늘 교체와 바늘이 손상된 경우 다시로드하거나, 흐름이 너무 강한 경우.되어야합니다 때로는 모세관 유리의 끝 부분이 폐쇄되고 어떤 흐름은 바늘에서 본 적이 있습니다. 이 경우 바늘을 교체하거나 조심스럽게 부드럽게 파열 바늘의 팁을에 문화 접시의 바닥에 그것을 낮추십시오. </li><li> 그것이 약 45 ° 각도로 세포쪽으로 향하고있다 있도록 바늘 끝을 위치. 세포쪽으로 이동하면서 다음 단칼에, 바늘을 낮추십시오. 두 번째 부드러운 모션과 함께, 즉시 세포에서 제거하기 위해 바늘의 방향을 반대로. </li><li> 성공적으로 주입 세포는 종종 약간 팽창하고 세포 내에 붉은 형광 rhodamine를 시각화하여 확인하실 수 있습니다. 때때로, 세포가 실수로 표시됩니다 핵에 주입됩니다. </li><li> 약 50-100 세포 주입 때까지 현미경의 단계를 조정하여 세포를 주입하기 위해 계속합니다. 현미경 스테이지를 이동하면, 그것이 세포의 상단을 지우는 있도록 microinjection 바늘을 제기해야합니다. </li></ol> 4. 대표 결과 : 시토크롬 C의 세포질 microinjection은 apoptosis 동안 mitochondria에서 출시된을 모방한 것이었 지요. 따라서, 예상대로, 섬유아 세포는 빠른 속도로 소 시토크롬 C (그림 1A)의 cytosolic microinjection에 apoptosis을 받고있다. 효모 시토크롬 C가 caspases 12 활성화의 능력이 있기 때문에 혼자 사출 절차 세포 죽음에 책임이 아니라는 것을 확인하려면, 효모 시토크롬 C의 주입은 중요한 조절 역할을합니다. 흥미롭게도, 사후 mitotic 교감 신경은 cytosolic 시토크롬 C (그림의 1B) 8,13 상당히 강한 있습니다. 우리 연구실은 내생 caspase 억제제의 XIAP는 뉴런 14 caspase 활성화의 핵심 억제제는 것을 확인했습니다. 따라서, 시토크롬 C 사출 다음 죽을 뉴런에, XIAP 먼저 inactivated가되어야합니다. 예를 들어, xiap에 시토크롬 C의 microinjection - / - 교감 신경이 세포에서 caspase 활성화 및 apoptosis (그림 2) 수 있도록 충분합니다. <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="그림 1"/> 그림 1. 시토크롬 C의 세포질 microinjection은 뉴런을 섬유아 세포에 신속하게 죽음을 유도하지만, 아닙니다. A) 와일드 타입 MEFs 또는 (B) 출생 후의 일 5 야생 형 교감 신경은 주입 세포를 표시하는 rhodamine - dextran과 함께 소 시토크롬 C (10 MG / ML)로 microinjected되었습니다. 이미지는 같은 셀 즉시 다음과 같은 사출 (0 HR) 분야, 또는 지정된 시간을 보여줍니다. 화살표가 주입된 세포를 나타냅니다. 스케일 바, 20 μm의. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="그림 2"/> 그림 2. XIAP - 결함 뉴런은 세포질 시토크롬 C의 microinjection에 감염될 수 있습니다. XIAP 녹아웃 마우스에서 출생 후의 5 일 동정 뉴런은 세포를 주입 표시 rhodamine - dextran과 함께 소 시토크롬 C (10 MG / ML)로 microinjected되었습니다. 이미지는 같은 셀 즉시 다음과 사출 분야 (0 HR) 또는 시토크롬 C의 microinjection 후 5 시간 (5 시간)를 보여줍니다. 스케일 바, 20 μm의.

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동물의 모든 실험 절차는 노스캐롤라이나 대학에서 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다.

직접 세포의 세포질에 시토크롬 C의 microinjection은 apoptosis의 포스트 mitochondrial 규제 연구 수있는 독특하고 강력한 도구입니다. 중요한 것은,이 기술은 셀룰러 또는 mitochondrial 손상을 에이전트를 사용하지 않고 mitochondria의 apoptosis 하류의 직접적인 활성화를 위해 수 있습니다. 이 프로토콜은 apoptosis에 대한 연구 시토크롬 C의 microinjection에 초점을 맞춘 반면, 여기?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> DM IRE2 인버티드 현미경 </td><td> Leica </td><td></td><td></td></tr><tr><td> PC - 10 Microinjection 니들 풀러 </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO - 202 Micromanipulator </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet Microinjector </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Microfilament와 얇은 벽 Boroscilicate 모세관 유리 </td><td> AM 시스템 </td><td> 615000 </td><td> 4 인치 길이, 1.00 mm 외경, 0.75 mm 내경 </td></tr><tr><td> Rhodamine B isothiocyanate - Dextran </td><td> 시그마 - 알드리치 </td><td> R9379 </td><td> 평균 분자량 70,000 ~ 다 </td></tr><tr><td> 보빈 시토크롬 C의 단백질 </td><td> 시그마 - 알드리치 </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

Apoptosis 또는 프로그램된 세포 죽음은 세포가 1 죽게하는 보존과 높은 규제 경로입니다. 세포가 세포 독성 스트레스의 다양한 발생하는 경우 Apoptosis가 발생하실 수 있습니다. 이러한 모욕 궁극적으로 mitochondrial intermembrane 공간에서 세포질 2 시토크롬 C의 릴리스의 원인이 신호 폭포를 시작합니다. mitochondria에서 시토크롬 C의 릴리스는 caspases, 궁극적으로 3-4 세포 죽음을 실행할 핵심 세포 프로 테아제의 급속한 활성화를 유발하는 핵심 이벤트입니다. apoptosis의 경로는 mitochondria 5 시토크롬 C 릴리스의 상류와 하류 지점에 규제입니다. caspase 활성화의 포스트 mitochondrial 규제를 공부하기 위해 많은 조사가 holocytochrome C (헴 연결된) 전지 6-9로 단백질의 직접적인 세포질 microinjection로 설정되어 있습니다. 시토크롬 C는 일반적으로 헴 그룹의 첨부 파일은 10-11 apoptosis 활성화하도록하는 데 필요한 어디 mitochondria에 언어입니다. 따라서 직접 caspases를 활성화하기 위해서는 cDNA 구조에서 시토크롬 C의 표현이 mitochondrial 타겟팅 및 헴 첨부집니다 동안 그것 cytosolic caspases에서 분리된 것입니다 때문에 holocytochrome C 단백질 대신 자사의 cDNA를 삽입하는 것이 필요합니다. 따라서 정화 헴 연결된 시토크롬 C 단백질의 직접 cytosolic microinjection은 셀룰러 및 mitochondrial 손상을 독성이 모욕을 사용하지 않고 mitochondrial 시토크롬 C 자료 및 apoptosis를 모방하는 유용한 도구입니다. 이 문서에서는, 우리는 마우스 배아 섬유아 세포의 (MEFs)와 예에서 볼 수 있듯이 기본 교감 신경을 사용하여 세포로 시토크롬 C 단백질의 microinjection을위한 방법을 설명합니다. 이 프로토콜은 apoptosis의 수사 시토크롬 C의 주입에 초점을 맞춘 반면, 여기에 나와있는 기술도 쉽게 관심을 다른 단백질의 microinjection에 대해 적용할 수 있습니다.

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

이 프로토콜에서는, 우리는 시토크롬의 직접적인 세포질 microinjection을 설명<em&gt; C</em섬유아 세포 및 기본 교감 신경으로&gt; 단백질. 이 기술은 시토크롬의 도입을 허용<em&gt; C</em세포 및 일일 시토크롬의 릴리스의 세포질에&gt; 단백질<em&gt; C</em&gt; mitochondria에서, 이것은 apoptosis 동안 발생합니다.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K 조회수.  University of North Carolina. 이 프로토콜에서는, 우리는 시토크롬의 직접적인 세포질 microinjection을 설명 C 단백질. 이 기술은 시토크롬의 도입을 허용 C 단백질 C mitochondria에서, 이것은 apoptosis 동안 발생합니다.

비디오: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

CD1d에 의해 Glycolipid의 항원 프레 젠 테이션 및 NKT 세포 활성화의 치료 가능성 : 인터뷰

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

연구

생물학

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus Laevis의 Oocytes의 Microinjection

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

트렌스 제닉의 생성 C. 엘레간스 Biolistic 변환에 의해

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Microinjection하여 포유 동물 세포에서 mRNA 원자력 수출 속도론 분석

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

편광 상피 세포에 Plasmids의 Microinjection에 의해 작은 GTPases의 기능을 분석

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

시토크롬를 사용 Mitochondrial 호흡 기능의 시각화 C 데이스 / 호박산 탈수소 효소 (COX / SDH)를 두 번 라벨 Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

의 합성<em&gt; 생체내에서</em&gt; MRI-감지 Apoptosis 프로브

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...