시그마 - 알드리치에 의해 투자

1. Mycoplasma 검출 <ol><li> 세포 배양 supernatants 직접 테스트하거나 예제는 나중에 사용하기 위해 준비하실 수 있습니다. 을 준비하기 위해 나중에 95에 살균 증폭 관과 부화에 뜨는 대신 100 μl를 사용 ° C 5 분. 일단 이것은 샘플이 최대 일주일 동안 2-8 ° C에 저장할 수 있습니다 완료됩니다. 직전 펠렛에 샘플 간단히 원심 분리기 (5 초) 모든 세포 파편을 실행합니다. </li><li> PCR에 대한 샘플을 준비하려면, 그 반응에 필요한 Jumpstart는 DNA 형성 촉매의 DNA 중합 효소 / rehydration 버퍼의 전체 볼륨을 결정합니다. 우리가 5 총 반응을 준비할 것이다. 다섯 번째 반응은 DNA 형성 촉매의 2.5μl 및 rehydration 버퍼의 114.5μl 필요합니다. 이것은 반응 및 시료 반응과 긍정적인 컨트롤에 대한 부정적인 제어 플러스 rehydration 버퍼의 24.5μl 당 rehydration 버퍼의 22.5μl 당 DNA 형성 촉매 중 하나 단위의 최소 포함됩니다. 반응마다의 DNA 중합 효소의 1 단위는 rehydration 버퍼의 해당 볼륨에 추가되어 있어야합니다. 이것이 사용되는 DNA 형성 촉매에 따라 달라질 수 있습니다. </li><li> 깨끗한 microcentrifuge 관에 DNA 형성 촉매의 DNA 중합 효소의 계산된 볼륨을 놓고 rehydration 버퍼의 계산 볼륨하십시오. 의 DNA 중합 효소 / rehydration 버퍼는 튜브를 flicking하여 부드럽게 혼합한다. 이 혼합물은 vortexed해서는 안됩니다. </li><li> 부정적인 제어를 준비하고 샘플 키트에 제공된 투명한 반응 튜브를 사용합니다. 키트에 제공된 반응 튜브는 이미 nuclueotides, primers 및 내부 제어 DNA가 포함되어 있습니다. 로 이전 단계에서 준비 Jumpstart는 DNA 형성 촉매의 DNA 중합 효소 / Rehydration 버퍼 믹스, 23 μl는 부정적인 제어 및 샘플 튜브의 각에 배치해야합니다. 부정적인 제어 DNA 무료로 물 2 μl를 추가하여 샘플 튜브 및 라벨의 각 샘플 2 μl를 추가합니다. 튜브를 flicking하여 내용을 섞는다. 내용은 vortexed해서는 안됩니다. </li><li> 준비하는 긍정적인 컨트롤은 키트에 제공된 핑크 반응 튜브를 사용합니다. 키트에 제공된 반응 튜브는 이미 nuclueotides, primers 및 내부 제어 DNA가 포함되어 있습니다. 반응 튜브 및 레이블로 이전 단계에서 준비 Jumpstart는 DNA 형성 촉매의 DNA 중합 효소 / Rehydration 버퍼 혼합 25 μl를 추가합니다. 튜브를 flicking하여 내용을 섞는다. 내용은 vortexed해서는 안됩니다. 대조군, 5 분 실온에서 긍정적인 제어 및 샘플 튜브를 품어. </li><li> PCR이 이루어 수있는 샘플은 열 자전거 타는 사람에 배치해야합니다. Jumpstart는 DNA 형성 촉매를 사용하면 정품 인증 단계는 필요하지 않습니다. 열 자전거 타는 사람의 반응 튜브를 놓습니다. 주기는 다음과 같이 설정되어야합니다 : 94 ° 30 초 동안 C, 55 ° 30 초 40초 72도 ° C에 대한 C. 이러한주기는 40 번 실행됩니다. </li><li> PCR주기가 완료되면 샘플은 열 자전거 타는 사람에서 그들을 제거하고 얼음에 그들을 배치하여 4-8 ° C로 냉각해야합니다. 샘플 냉각있을 때 그들은 전기에 대한 아가로 오스 겔에로드해야합니다. 그들은 이미 반응 튜브에 존재로서 로딩 젤 및 염료는 필요하지 않습니다. 직접 별도의 차선으로 각 PCR 8 μl를로드합니다. 3.0 cm - 2.5 마이 그 레이션 후 전기를 중지합니다. </li></ol> 2. 대표 결과 대조군은 약 481 BP에서 밴드를 표시해야합니다. 이것은 비디오의 시작 부분에 표시되는 전기 영동 젤에 대한 부정적인 컨트롤 열에 볼 수 있습니다. 481 BP의 부정적인 제어 밴드의 부재는 효소의 활동이 sufficienttaq만큼 민감한 아니었 사용되지 않았음을 좋은 지표입니다. Mycoplasma 긍정적인 샘플 270의 범위에서 밴드 ± 8 BP를 표시합니다. 밴드는 고도로 오염된 샘플 무겁고 가벼운 오염 샘플 희미합니다. 강도의 차이는 비디오의 시작 부분에 표시되는 전기 영동 젤에 긍정적인 샘플 열에 볼 수 있습니다. 모든 샘플은 PCR이 예상대로 발생는 것을 입증하는 내부 대조군을 포함합니다. 그것은 매우 오염된 샘플에 대한 내부 통제의 부재를보고 정상입니다. 샘플에 대한 부정적인 범위에서 긍정적인 범위 또는 밴드에서 밴드가 없다면 이것은 샘플 저해하는 좋은 지표입니다. 샘플을 저해하는 경우 PCR 억제제는 DNA 추출을 수행하여 제거할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3057/3057fig1.jpg" /> . 그림 1 관련 Amplicon 밴드 : PCR에 의해 mycoplasma 검출 결과가 표시됩니다. 레인 2는 약 481 BP에서 밴드를 표시해야 대조군입니다. 481 BP의 부정적인 제어 밴드의 부재는 DNA 형성 촉매 사용이 충분 구분하지다고 좋은 지표입니다. Mycoplasma - 긍정적인 샘플 270의 범위에서 밴드를 표시합니다 +로 차선 3과 같이 8 BP - 6. 밴드 hea 것입니다VY 매우 오염된 시료 (레인 6)와 가볍게 오염된 시료 (레인 4)을 위해 만들어졌습니다. 모든 샘플은 PCR이 예상대로 발생는 것을 입증하는 내부 대조군 (481 BP)를 포함하고 있습니다. 그것은 매우 오염된 샘플에 대한 내부 통제의 부재를보고 정상입니다.

<ol>
	<li>Wirth, M., Hauser, H., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+Sensitivity+of+Polymerase+Chain+Reaction+(PCR)+in+Comparison+with+Other+Methods+for+the+Detection+of+Mycoplasma+Contamination+in+Cell+Lines.">Specificity and Sensitivity of Polymerase Chain Reaction (PCR) in Comparison with Other Methods for the Detection of Mycoplasma Contamination in Cell Lines.</a> Journal of Immunological Methods. 164, 91-100 (1993).</li><li>Kong, H., Volokhov, D. V., George, J., Ikonomi, P., Chandler, D., Anderson, C., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+Cell+Culture+Enrichment+for+Improving+the+Sensitivity+of+Mycoplasma+Detection+Methods+Based+on+Nucleic+Acid+Amplification+Technology+(NAT).">Amplification of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT).</a> Applied Microbiology and Biotechnology. 77, 223-232 (2007).</li><li>Volokhov, D. V., Graham, L. J., Borson, K. A., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+Testing+of+Cell+Substrates+and+Biologics:+Review+of+Alternative+Non-Microbiological+Techniques.">Mycoplasma Testing of Cell Substrates and Biologics: Review of Alternative Non-Microbiological Techniques.</a> Molecular and Cellular Probes. , (2011).</li></ol>

저자는이 문서에서 사용하는 시약 및 도구를 제작 시그마 - 알드리치의 직원입니다.

때문에 키트의 극단적인 감성, 정확하게 수행하면 키트 샘플은 mycoplasma 오염을 가지고 있는지 여부를 나타내는 정확한 결과를 얻을 수 있습니다. 키트의 Jumpstart DNA 형성 촉매의 DNA 중합 효소와 함께 사용하기위한 최적화되었습니다. 기타 taqs는 샘플을 준비할 때 사용할 수 있지만 481 BP의 내부 통제 밴드는 대체 DNA 형성 촉매의 적절한 민감도를 나타내기 위해 존재해야합니다. 긍정적인 밴드가 오염의 수준에 따라 다양한 밀도를 전시하는 것은이 가능합니다. 많이 오염된 샘플에 대한 내부 통제 대역의 부재를 관찰하는 것이 가능합니다. 오염을 방지하기 위해 샘플을 준비 때는 주의해야하며 적절한 기술을 사용하면 적절한위한 열쇠입니다. 룩아웃 Mycoplasma PCR 감지 키트를 사용하여 감지.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 룩아웃 Mycoplasma PCR 감지 키트 </td><td> 시그마 - 알드리치 </td><td> MP0035 </td><td> </td></tr><tr><td> Jumpstart를 DNA 형성 촉매의 DNA 중합 효소 </td><td> 시그마 - 알드리치 </td><td> D9307 </td><td> </td></tr><tr><td> GenElute 혈액 게놈의 DNA 키트 </td><td> 시그마 - 알드리치 </td><td> MP0030 </td><td> </td></tr><tr><td> Nunc 증폭 튜브 </td><td> 시그마 - 알드리치 </td><td> T0447 </td><td> </td></tr></tbody></table>

optimized pcr-based detection of mycoplasma

오염없는 세포 라인의 유지 보수 세포 기반 연구하는 것이 필수적입니다. 가장 큰 오염 문제 중 mycoplasma 오염 수 있습니다. 일반적으로 오염된 세포를 mycoplasma 죽이지 아니지만, 그들이 감지하기 어려울 수 있으며 변경 신진 대사를 포함하여 교양 세포에 효과 다양한 발생할 수, 증식 및 염색체 aberrations가 둔화. 즉, mycoplasma 오염은 생명 과학 연구에 대한 정확한 자료를 제공하는 이들 세포 라인의 가치를 타협. 실험실에서 mycoplasma 오염의 원인은 매우 완벽하게 제어하는​​ 도전입니다. 특정 mycoplasma 수종은 인간의 피부에 발견으로, 그들은 가난한 무균 기술을 통해 도입하실 수 있습니다. 또한, 그들은 이러한 태아 소 혈청으로 오염된 보충제에서 온 수 있으며, 가장 중요한 기타 오염된 세포 문화. 일단 mycoplasma가 문화를 오염, 빨리 연구실의 다른 영역을 오염으로 확산될 수 있습니다. 좋은 무균 기술로 좋은 실험실 관행에 엄격한 준수가 핵심이며, mycoplasma에 대한 정기 검사는 높은 mycoplasma 오염의 성공적인 제어를위한 권장합니다. mycoplasma의 PCR 기반의 감지는 일상적인 세포 라인 유지 보수를 위해 매우 인기있는 방법이되었습니다. PCR 기반의 탐지 방법은 매우 민감하고 연구자들이 미생물 기술을 사용하여 필요한 그것이 시간에 비해 감지되면 오염을 분리 및 제거에 신속하게 대응할 수 있도록 신속한 결과를 제공할 수 있습니다. 룩아웃 Mycoplasma PCR 탐지 키트 μl 당 겨우 2 genomes의 검출 한도, 매우 민감합니다. 매우 구체적인 Jumpstart는 DNA 형성 촉매의 DNA 중합 효소와 독점적인 프라이머 설계의 이점 복용, 잘못된 반응을 획기적으로 줄일 수 있습니다. 편리한 8 튜브 형식은, dNTPs 사전 - 코팅 및 관련 primers 스트립은 세포 라인의 큰 컬렉션과 함께 고객의 요구를 충족하기 위해 처리량을 높일 수 있습니다. 키트의 극단적인 감도 감안할 때, 큰주의가 샘플 및 시약의 실수로 오염을 방지하기 위해 이동해야합니다. 우리가 보여주는 단계별로 프로토콜은 신뢰할 수있는 mycoplasma 감지에 필요한주의 사항과 관행을 강조 표시합니다. 우리는 또한 표시하고 전형적인 결과와 자신의 해석을 논의합니다. 우리의 목표는 룩아웃 Mycoplasma PCR 감지 키트를 사용하여 연구의 성공을 보장하는 것입니다.

Watch this Scientific Journal Video about Optimized PCR-based Detection of Mycoplasma at JoVE.com

Optimized PCR-based Detection of Mycoplasma

룩아웃 Mycoplasma PCR 탐지 키트는 세포 문화와 생체를 파생 다른 세포 배양에서 Mycoplasma Acholeplasma, 그리고 Ureaplasma 오염의 검출에 높은 민감도에 대한 선택의 방법으로 설립되는 효소의 연쇄 반응 (PCR)을 사용합니다.

Mycoplasma의 최적화된 PCR 기반의 감지

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

optimized-pcr-based-detection-of-mycoplasma

Research

JoVE Journal

Biology

53.5K 조회수.  Sigma-Aldrich. 룩아웃 Mycoplasma PCR 탐지 키트는 세포 문화와 생체를 파생 다른 세포 배양에서 Mycoplasma Acholeplasma, 그리고 Ureaplasma 오염의 검출에 높은 민감도에 대한 선택의 방법으로 설립되는 효소의 연쇄 반응 (PCR)을 사용합니다.

비디오: Optimized PCR-based Detection of Mycoplasma

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis 연구에 최적화된 섬유소 젤 비드 분석

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

연구

생물학

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Zymography의 기능 매트릭스 Metalloproteinases의 감지

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

리어링을위한 최적화된 프로토콜 Fopius arisanus Tephritid 과일 Parasitoid 난다

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

쥐 모델에 최적화 된 구조를 가진 Electrospun 혈관 이식 술의 이식

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

체 외에서 세포 마이그레이션 자동화 된 광학 카메라의 테스트에 대 한 스크래치 분석 결과 최적화

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

면역 형광에 의한 1 차 적인 섬모의 간단한 검출

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

엑소시토시스의 자동 검출 및 분석

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...