1. lipids 준비 <ol><li> 이중층의 상단에 전단지에 단백질을 연결하기 위해, 우리는 니켈 2를 사용 + 폴리 - 히스티딘 태그로 재조합 단백질을 고정 chelating의 lipids. 특정 세포 유형 니켈 2 + - 코팅 bilayers 이외의 특별히 준수지만, T - 세포는 일반적으로 T 세포 활성화를 공부하고이 방법이 최적 만들고하지 않습니다. </li><li> 지질 이중층 솔루션은 20% 니켈 2 솔루션을 준비하여 만든 + chelating의 지질 및 클로로포름 - 메탄올 1-2 ML있는 유리관에서 해당 금액의 80 % phosphatidylcholine. <ol><li> 따뜻한 (30-37 ° C) 물을 욕조에서 질소 가스 스트림에서 용매를 증발. 이것은 일반적으로 십오분 주위에 걸립니다. </li><li> 90 분간 lyophilizer을 사용하여 고진공 하에서 남아있는 용매를 제거합니다. </li></ol></li><li> 2퍼센트 혼합 세제 / 인지질 micelles의 솔루션을 만듭니다 트리스 - 생리 버퍼에 N - octyl - glucoside 세제에 lipids를 해산. 최종 인지질의 농도는 0.4 MM해야합니다. liposomes을 형성하기 위해 세제 및 양식 liposomes를 제​​거하는 트리스 - 살린 3 변경 사항에 대해이 솔루션을 dialyze. 우리는 보통 10 KDA이 떨어져 상처와 6mm 직경 스펙트럼 / 포어 # 2 튜브 1 개 ML의 순서에 볼륨과 함께 작동합니다. 우리는 튜브를 클램핑 때 어떤 공기 방울의를 제외주의합니다. 우리는 일반적으로 무균 필터이 솔루션 70 %의 에탄올 소독 장갑을 사용하여 처리하고 깨끗한 조건에서 투석을. </li><li> 이 솔루션은 수정 같이 맑은해야합니다. 그것은 산화를 방지하기 위해 아르곤 가스를 아래에 저장됩니다. 솔루션 흐린있다면, 다중 벽으로 둘러싸인 vesicles가 생성되었습니다, 당신은 다시 시작해야합니다. </li></ol> 2. ICAM - 1 및 MHC가 평면 이중층에 예금 얼마나 많은 결정 <ol><li> 실험을 설정하기 위해서는 ICAM - 1 및 MHC는 수용성 단백질의 특정 농도에 + chelating 이중층 니켈 2 주어진 표면 영역에 입금이 어느 정도 결정 먼저 필요합니다. 이렇게하려면, 예금 bilayers 5 마이크로 미터 직경의 유리 비즈에서 조건 하에서 단백질 솔루션 유리 비즈를 품어 그 대략적인 이미징에 사용되는 흐름 세포에 해당하고, 흐름 immunofluorometric 분석에 의해 바운드 단백질의 밀도를 읽어보십시오. </li><li> 플루오레신 당 분자 알려진 숫자 FITC - 표시된 항체는 표면 바운드 단백질을 감지하는 데 사용됩니다. 플루오레신 표준 구슬은 흐름 microfluorimeter을 교정하는 데 사용됩니다. </li><li> 우리는 조건을 확립 것을 대략 이들에 대한 항원 제시 ICAM - 1 200 molecules/μm2 및 I - EK - MCC91 - 103 복합 0.2-20 molecules/μm2와 세포. </li></ol> 3. 평면 bilayers 형성을위한 유리 커버 전표 청소 <ol><li> 평면 bilayers은 liposomes 유리에 퓨즈 때 저절로 형성하지만 유리가 깨끗한지 경우에만. </li><li> 피라 냐가 작업 때, 우리는 산성 앞치마와 무거운, 산성 모성 장갑을 포함한 전체 보호복을 착용하십시오. 조심스럽게 유기 폐기물에서 폐기물을 분리하고 적절하게 처리. </li><li> 산성 피라 냐가 솔루션을 준비하려면, 마른 비커에 농축 황산의 75 ML을 추가하고, 신중하게 30 % 과산화수소 25 ML를 추가합니다. 이 솔루션은 빠른 속도로 이상 100까지 가열 ° C. </li><li> 15 분 용액의 건조 coverslips 젖어. 솔루션에서 제거하고 양쪽에 잠시 물을 정화에 린스. 정화 물을 배수하기 위해 진공 소스를 사용하여 coverslips 건조. 피라 냐가 솔루션 괜찮아, 잘 퓸 후드에 표시된 캡 느슨한와 남아있는 피라 냐가 폐기물 컨테이너에 추가할 수있게 해줍니다. liposomes, coverslips, 그리고 단백질의 모든 준비가되면 면역 버렸네가 형성 될 준비가 된 것입니다. </li></ol> 4. bilayers을 형성 <ol><li> bilayers을 형성하기 위해 우리 실험실 통합 난방의 장점을 가지고 Bioptechs FCSII 회의소를 사용합니다. FCSII 시스템은 스테인레스 스틸베이스입니다 함께 클램프 반대편에 유체 홈과 0.5 mm 최고 가스켓 하나의 표면에 가열을위한 인듐 주석 산화물 코팅 microaqueduct 슬라이드와 microfluidic 매니폴드, 먼지없는 0.25 mm 흐름 챔버와 피라 냐가의 높이를 정의 가스켓은 이중층이 형성되는 40mm의 원형 coverslip 청소. </li><li> 이중층이 형성되는시 커버 슬립이 높은 친수성과 청소해야 할 동안 더 소수성의 경우, microacqueduct 슬라이드 실제로 잘 작동합니다. microaqueduct보다 소수성이 다음 깨끗한 물로 세척하고 완전히이 컨디셔닝 과정 후에 건조뿐만 사용 사이로 실온에서 60 분 1 % HSA로 치료에 의해 이루어집니다 만들기. 이 절차에 의해 왼쪽 변성 단백질의 코팅 리포좀 현탁액 1 μl는 원형, hemispherica를 형성하실 수 있습니다&gt; 0.25 mm 높은 내 놓습니다. </li><li> 평평한 표면 위에, 위쪽 감독 튜브, 흐름 세포 및 양식 평면 bilayers 조립 매니폴드를 배치합니다. 그런 다음 0을 넣으십시오. 그것이 평면 앉아 있는지 확인하고, 유체 튜브를 통해 위치에 구멍 5mm 개스킷. 부드럽게까지 직면하고있는 유체 채널과 개스킷에 microaqueduct 측면을 배치하고 슬라이드에 구멍이 microfluidic 튜브 정렬 것을 확인하기 전까지는 모든 하향 압력을 적용하지 않고 평면 좌석 있는지 확인하십시오. 채널은 유체 채널로 줄지어 않으며 주요 공기 공간이 없습니다되도록 microaqueduct 슬라이드의 위에 밖으로 직사각형 절단과 먼지없는 0.25 mm 개스 라이. </li><li> 각 드롭 사이의 중심 - 투 - 중심 거리 2.5 mm는 그러한 패턴에 microaqueduct 슬라이드의 표면에 5 리포좀 정지 X 1 μl 방울을 넣으십시오. 5 리포좀 방울 다른 작곡있을 수 있지만,이 경우 우리는 두 bilayers, 아니 니켈이 하나의 + + chelating의 lipids와 10 % 니켈 2 + + chelating lipids의 한.을 형성합니다 </li><li> 이 방울이 자리에되면, 신중하게 커버 한 움직임의 표면에 아래로 살짝 놓습니다. 가능한 한 빨리 기지로 클램프. 리포좀 방울 커버 슬립과 접촉을해야합니다. bilayers가 이미 형성 이후이 연락처는 부러진해서는 안됩니다, 모든 장애는 결함이 bilayers가 발생할 수 있습니다. </li><li> 현미경에 coverslip의 위치를​​ 돕기 위해 microacqueduct 슬라이드에 몇 가지 작은 잉크의 명소로 bilayers의 위치를​​ 표시하기 위해 기억하는 것이 중요합니다. 시스템이 equilibrated이 있는지 확인하기 위해 20 분이 걸립니다. 20 유연한 튜브의 cm와 3 방향 꼭지에 연결된 20 ML의 주사기를 사용하여 모든 솔루션을 전송합니다. 꼭지와 흐름 세포 사이의 죽은 볼륨을 최소화하기 위해 튜브의 짧은 길이에 의해 흐름 세포에 꼭지의 다른 포트를 연결합니다. 이 MM MgCl 2, 1 MM CaCl 2, 5 MM 포도당, 1 % 인간의 혈청 알부민을 포함한 생리 HEPES - 버퍼와 주사기를 채우십시오. </li><li> 기포가가되지 않도록 프라임 관 그리고 꼭지. 흐름 세포에 이것을 연결하고 공기 방울이 이중층을 통해 전달하지 않도록 보면서 한 동작에 5 ML 통해 밀어. 지금, 당신은 bilayers을 것입니다. </li></ol> 작성자 :이 동영상이 문서는 &quot;Amoeboid 로코의 테마 유사 면역 시냅스와 Kinapses 채취와 위로 헌터&quot;을 지원 <a href="http://arjournals.annualreviews.org/action/doSearch?action=runSearch&amp;type=advanced&amp;result=true&amp;prevSearch=%2Bauthorsfield%3A%28Dustin%2C+Michael+L.%29" target="_blank">마이클 L. 더스틴</a> , <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.24.110707.175226">세포 및 발달 생물학의 연례 검토</a> , 볼륨 24, 2008.

supported planar bilayers for the formation of study of immunological synapses and kinapse

지원되는 평면 bilayers는 면역 버렸네에서 분자 상호 작용을 모델로 사용할 수있는 강력한 도구입니다. lymphocytes와 항원 제시 세포 간의 상호 작용을 모방하기 위해, 우리는 폴리 - 히스티딘 태그 이중층의 위쪽 전단지에 재조합 세포 접착 및 MHC 단백질을 앵커로 Ni2 + - chelating의 lipids를 사용합니다. 평면 bilayers은 지방질을 준비 이중층이 형성 유리 표면을 처리하고, 다음 lymphocytes가 추가됩니다 흐름 세포를 포함하는 전문 챔버의 이중층을 형성하여 생성됩니다. 그런 다음, bilayers은 니켈과 그의 - 태그 재조합 단백질로 청구됩니다이 추가됩니다. 마지막으로, lymphocytes은 흐름 세포와 TIRF 현미경으로 주사는 이미지 버렸네 형성과 구조하는 데 사용할 수있는 제어하는​​ T 세포의 운동, 수용체 정렬의 사이트 및 수용체 저하의 사이트.

Watch this Scientific Journal Video about Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse at JoVE.com

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

지원되는 평면 bilayers는 면역 버렸네에서 분자 상호 작용을 모델로 사용할 수있는 강력한 도구입니다. 여기, 우리는 지질 bilyer의 위쪽 전단지에 버렸네 형성을 조절하고 TIRF 현미경을 사용하여 버렸네 형성을 시각화하는 것으로 알려진 세포 부착 단백질을 고정하는 방법을 보여줍니다.

면역 시냅스와 Kinapse 연구의 형성을위한 지원 평면 Bilayers

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

supported-planar-bilayers-for-formation-study-immunological-synapses

Research

JoVE Journal

Biology

12.5K 조회수.  New York University - NYU. 지원되는 평면 bilayers는 면역 버렸네에서 분자 상호 작용을 모델로 사용할 수있는 강력한 도구입니다. 여기, 우리는 지질 bilyer의 위쪽 전단지에 버렸네 형성을 조절하고 TIRF 현미경을 사용하여 버렸네 형성을 시각화하는 것으로 알려진 세포 부착 단백질을 고정하는 방법을 보여줍니다.

비디오: Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

전기 생리학 실험을위한 인공 Bilayers의 준비

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Zebrafish 상악 바벌의 연구 방법

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

의 분리 및 정제 Drosophila</em마그네틱 비드별로 정렬> 주변 뉴런

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

에 대한 내피 세포의 관 형성의 분석<em&gt; 체외</em&gt; 혈관 신생 연구

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

면역 시냅스와 Kinapse 연구의 형성을위한 지원 평면 Bilayers

요약

더 많은 비디오 탐색

이 비디오의 챕터

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

컴퓨터가 만든 동물 모델 자극

전기 생리학 실험을위한 인공 Bilayers의 준비

Zebrafish 상악 바벌의 연구 방법

의 분리 및 정제 Drosophila 주변 뉴런

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (TED)

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

에 대한 내피 세포의 관 형성의 분석

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사용자 친화적 인 소프트웨어 응용 프로그램

면역 시냅스와 Kinapse 연구의 형성을위한 지원 평면 Bilayers

요약

더 많은 비디오 탐색

이 비디오의 챕터

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

컴퓨터가 만든 동물 모델 자극

전기 생리학 실험을위한 인공 Bilayers의 준비

Zebrafish 상악 바벌의 연구 방법

의 분리 및 정제 Drosophila 주변 뉴런

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (TED)

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

에 대한 내피 세포의 관 형성의 분석

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사용자 친화적 인 소프트웨어 응용 프로그램