이 작품은 퐁 드 물리 쉬르 라 자연 동부 표준시 레 기술 드 퀘벡, 혁신 및 보건 연구 (CIHR)의 캐나다 연구소에 대한 캐나다 fundation에 의해 투자되었다.

1. 프로브 헤드 (동영상에 표시되지 공정)의 Microfabrication <ol><li> 1 μm의 두께 열 SiO2 레이어와 525 μm의 두께가 네이 웨이퍼, 직경 사인치은, 4000 RPM 45의를위한 포토 레지스트 (PR)와 spincoated 있습니다. </li><li> 웨이퍼 50 s에 대한 110C에서 prebaked 및 개발 DI에 씻어서 5 S에 대한 모든 요소 (포트와 microchannels)을 갖춘 마스크를 통해 노출됩니다. </li><li> 덮개 SiO2가 1시 7분에 거리에 새겨져 것은 ≈ 15 분 (SiO2가 에칭되어 기판의 dewetting 에칭의 완성을 나타냅니다)에서 불화 수 소산​​ 산성 (BHF)이 솔루션 버퍼. O2 플라즈마이나 아세톤이 재가하는 데 사용하거나 남은 PR을 스트립입니다. </li><li> 두 번째 PR 층은 스핀 코팅 ≈ 10 μm의 [31]의 두꺼운 overlayer을 항복, 45 s에 대한 1,500 rpm으로. 이 PR 층 아래 SiO2 패턴은 아직 볼 수 있으며 포트는 특징으로 보조 마스크와 웨이퍼를 정렬하는 데 사용됩니다. </li><li> 노출과 PR의 개발 후, 웨이퍼는 씻어서 건조, 20 분 95C에 postbaked입니다. </li><li> 시 웨이퍼는 척을 보호하기 위해 녹인 흰색 왁스와 지원 웨이퍼에 연결되어 있습니다. </li><li> 유도 결합 플라즈마 (ICP) 드리이 3 단계 과정에서 웨이퍼 지형에 SiO2 패턴을 PR를 전송하는 데 사용 및 임베디드입니다 <ol><li> 드리이시 (두꺼운 PR에 의해 정의된 패턴)에 ≈ 500μmdeep 포트를 만들 수 있습니다. </li><li> 드리 시스템에서 웨이퍼를 언로 드하지 않고, PR은 플라즈마를 사용하여 재를 떨어서 있습니다. </li><li> 노출 SiO2 패턴은 50 μm의 깊이 채널을 생성하고, 웨이퍼를 통해 충전 및 방출 포트를 열고, 두 번째 드라이 에칭 프로세스에 대한 마스크 역할을합니다. 하역 후, 지원 웨이퍼는 온수의 흐름에 따라 분리됩니다. micromachined 웨이퍼는 다음 아세톤, 에탄올과 DI와 함께 청소합니다. </li></ol></li><li> 개별 MFP 칩이 절단됩니다. </li><li> PDMS 인터페이스 블록은 두 구조 폴리 (methylmethacrylate) (PMMA) 요소, 바닥을 형성 광택 강판, 두 개의 모세 혈관 (각각 두 비아스 액세스 중 하나에 삽입 구멍 인들로 구성된 micromould로 캐스팅하여 가공합니다 강판)은 유체 연결 구멍 장소 소유자로 재직. PDMS는 최소한 1 H. 위해 60C에 오븐에 완치됩니다 </li><li> PDMS 블록 230W 24의 1 mbar에서 공기 플라즈마의 두 부분을 활성화하고, 집에서 만든 기계 정렬 원조를 사용하여 두 가지를 가입하여 절단 MFP 실리콘 칩에 접착됩니다. </li><li> 어셈블리는 1 시간의 최소 60C 오븐에 공채로 남아 있습니다 </li></ol> 2. MFP의 어셈블리 <ol><li> 가스 기밀 유리 주사기는 기포가 존재하지 않습니다 않도록 플라스틱 주사기와 바늘을 사용하여 적절한 시약으로 가득합니다. 일반적으로, 우리는 주사에 대한 1 -10 microliter의 주사기와 열망에 대한 50-10 배 더 큰 볼륨과 주사기를 사용합니다. </li><li> 주사기가 낮은 죽은 볼륨 Nanotight 피팅을 사용하여 튜브를 모세관에 연결되어 있습니다. </li><li> 모세 혈관은 자필 현미경으로 거품을 확인합니다. </li><li> MFP 칩은 모세 혈관을 연결할 때 거품의 트래핑 방지하기 위해 버퍼 솔루션 prefilled입니다. </li><li> 모세 혈관은 프로브 헤드에있는 PDMS 연결 부분에 전원 연결 </li></ol> 3. 설정 MFP의 <ol><li> 프로브 헤드는 프로브 홀더에 고정되어 있으며 거꾸로 현미경 꼭대기 프로브 스테이션에 장착할 수 있습니다 </li><li> 주사기는 고정밀 주사기 펌프에 게재됩니다. </li><li> 이러한 유리 슬라이드로 기판은, 현미경 단계에 부착된있는 집에서 만든 홀더에 삽입됩니다. </li><li> MFP의 메사와 기판의 병렬는 MFP가 기판에 접촉 반입 때 나타나는 뉴턴의 반지 (간섭 변두리) 관찰에 의해 goniometers 한 켤레를 사용하여 조정됩니다. 연락처 및 반지의 주파수의 포인트는 기울기의 표시로 제공하고 있습니다. MFP가 표면에 부합되면, 하나의 간섭 반지는 전체 표면을 통해 확장됩니다. 이 법안은 또한 MFP와 기판 사이의 간격을 보정하는 역할을합니다. </li><li> MFP와 기판 사이의 간격은 표면 patterning 공정에 대한 중요합니다. 기판이 MFP 아래를 검색하여 처리되기 때문에, 수평 정렬은 마이크로 미터의 정밀도로 조정해야 세 마이크로 미터 나사에 의해 형성된 쓰리 포인트 지원을 사용하여 이루어집니다 있습니다. </li></ol> 4. MFP의 운영 <ol><li> 분배는 LabView 소프트웨어를 통해 제어됩니다. 장치 작업이 안구와 CCD 카메라를 사용하여 시각입니다. 사출 : 흡인 비율은 주변 버퍼와 원하는 기하학적 흐름 패턴과 시약의 확산에 따라 1시 3분에서 1시 10분 다릅니다. </li><li> 흡인의 주사기와 거품의 존재를 제대로 작동 확인하려면 먼저 적절한 열망을 시작하기 전에 흡인의 주사기로 액체를 주입.</li><li> 액체와 구슬이나 형광등 추적 염료의 모니터 흐름과 구속의 분사를 시작합니다. </li><li> 특정 응용 프로그램의 프로브를 사용하여, 즉 에칭 또는 표면이나 세포의 얼룩, 증착을위한 표면 처리를 통해 검색합니다. </li></ol>

<ol>
	<li>Juncker, D., Schmid, H., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature Materials. 4 (8), 622-622 (2005).</li></ol>

이 시약 및 기판의 종류와 함께 사용 (2) 적응, (I) 휴대이며 (3) 큰 영역을 통해 운영 할 수 있기 때문에 microfluidic 프로브 (MFP)는 다목적입니다. 불필요한 거품이 거품을 방지하기 위하여 흐름의 중단을 초래할 수있는 모든 구성 요소가 조립하기 전에 액체로 가득해야합니다. 프로브와 표면 사이의 간격은 몇 micrometers입니다 아직 메사 수백 micrometers 폭이며, cm의 범위에 거리가 ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>microfluidic connectors</td>
<td>&nbsp;</td>
<td>Upchurch Scientific</td>
<td>Micro- and Nano-tight fittings and sleeves</td>
<td>&nbsp;</td>
<tr>
<td>2-component manual dispenser</td>
<td>&nbsp;</td>
<td>Conprotec Inc.</td>
<td>DM400</td>
<td>To dispense and mix PDMS mixture</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Version 8.0</td>
<td>&nbsp;</td>
<tr>
<td>Mechanical Convection Oven</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>1330FM</td>
<td>&nbsp;</td>
<tr>
<td>Glass syringes</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>&nbsp;</td>
<td>Polymicro Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasma Chamber</td>
<td>&nbsp;</td>
<td>Tegal Corporation</td>
<td>Plasmaline 415</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>TE2000-E</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pumps</td>
<td>&nbsp;</td>
<td>Cetoni</td>
<td>neMESYS</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
<tr>
<td>Camera</td>
<td>&nbsp;</td>
<td>Photometrics</td>
<td>QuantEM 512SC</td>
<td>&nbsp;</td>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microfluidic probe holder goniometers</td>
<td>&nbsp;</td>
<td>Melles-Griot</td>
<td>07GON504</td>
<td>&nbsp;</td>
<tr>
<td>Linear stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>LS-50</td>
<td>For z-control of the MFP</td>
</tr>
<tr>
<td>Manual linear stage</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>443-4 Series</td>
<td>For x- and y- axis control of the MFP</td>
</tr>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>PZ-2000</td>
<td>With x-, y- and z- control</td>
</tr>		</tbody>
		</table>
		</div>

the microfluidic probe: operation and use for localized surface processing

Microfluidic 장치는 assays이 샘플 미세한를 사용하여 수행할 수 있도록 최근 세포의 microenvironment를 제어하는​​ 데 사용되었습니다. Microfluidics은 일반적으로 한정된 볼륨 내에 도입하고, 세포의 경우 교양 수있는 샘플을 자신의 사용을 제한 폐쇄 microchannels와 연결되어 있습니다. 한편, 시스템을 micropipetting 것은 특히 소스와 싱크 등 다른 하나지만, 흐름의 감금으로 하나 피펫의 작동은 3 차원에 어려운 푸시풀 설정을 사용하여 로컬 세포 표면 perfuse하는 데 사용되었습니다. 또한, pipettes는 불안정하고 어려운 위치이며, 따라서 오직 정적 구성에 사용됩니다. microfluidic 프로브 (MFP)는 폐쇄 microfluidic 채널의 구축에 의해 부과하고 대신 microfluidic 시스템에 샘플을 둘러싸고의 microfluidic 흐름이 직접 시료에 전달 될 수 있으며, MFP를 사용하여 샘​​플을 통해 스캔한 제약을 circumvents. . 간격으로 주입 microjet은 주변 액체의 유체 힘에 의해 갇혀과 완전히 다른 오프닝으로 다시 aspirated되도록 분사와 열망 오픈은 서로 micrometers 몇 수십 내에 위치하고 있습니다. microjet는 기판 표면에 걸쳐 플러시 및화된 증착 / 표면에 걸쳐 탐사를 검색하여 큰 영역을 통해 사용할 수있는 시약의 전달을위한 정밀한 도구를 제공하실 수 있습니다. 이 비디오에서 우리는 microfluidic 프로브 1 (MFP)를 제시한다. 우리는 MFP를 조립하는 방법을 자세히 설명, 거꾸로 현미경 위에 그것을 마운트하고, 기판 표면에 상대를 정렬하고, 마지막으로 버퍼에 포장되어 기판 표면을 처리하는 데 그것을 사용하는 방법을 보여줍니다.

Watch this Scientific Journal Video about The Microfluidic Probe: Operation and Use for Localized Surface Processing at JoVE.com

The Microfluidic Probe: Operation and Use for Localized Surface Processing

이 비디오에서 우리는 microfluidic 프로브를 제시<sup&gt; 1</sup&gt; (MFP). 우리는 MFP를 조립하는 방법을 자세히 설명, 거꾸로 현미경 위에 그것을 마운트하고, 기판 표면에 상대를 정렬하고, 마지막으로 버퍼 용액에 잠겨 기판 표면을 처리하는 데 그것을 사용하는 방법을 보여줍니다.

Microfluidic 프로브 : 운영 및 현지화된 표면 처리에 사용

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

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Biology

8.8K 조회수.  McGill University. 이 비디오에서 우리는 microfluidic 프로브를 제시 1 (MFP). 우리는 MFP를 조립하는 방법을 자세히 설명, 거꾸로 현미경 위에 그것을 마운트하고, 기판 표면에 상대를 정렬하고, 마지막으로 버퍼 용액에 잠겨 기판 표면을 처리하는 데 그것을 사용하는 방법을 보여줍니다.

비디오: The Microfluidic Probe: Operation and Use for Localized Surface Processing

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

현지화된 RNAi과 약용 거머리의 이소성 유전자 발현

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

연구

생물학

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

자기편 셀룰러 문화에 대한 산소 삽입 제작 및 운영

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

계면 세포 핵의 기계적 성질을 알아내기 위해 Biophysical Assays : 기판 스트레인 신청 및 Microneedle 조작

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

미생물 시스템 생물학의 Chemostats의 사용

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

마우스 유선 화 된 약물 전달을위한 췌관 주입

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

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

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

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

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

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

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

말린된 피 명소-준비 및 Immunoassays 및 분자 기술 사용에 대 한 처리

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

마이크로 유체 장치를 사용하면 단일 신진 효모 세포의 수명과 세포 표현형을 측정하는

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

마우스에 부분적 담 관 결 찰: 지역화 된 폐쇄성 Cholestasis의 제어 모델

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...