원자력 과학 및 공학의 호주 연구소의 지원 인정받고 있습니다. TCK는 AINSE 보너스받는했다. Epigenomic 의학 연구소는 국민 건강과 호주의 의료 연구위원회 (566559)에 의해 지원됩니다. HR은 대학원 호주와 BakerIDI 밝은 점화 수상에 의해 지원됩니다. 이 작품은 바이오 메디컬 이미징 개발 회사 (CRC - 입찰가) 설립과 호주 정부의 협동 연구 센터 프로그램에 따라 지원을 위해 CRC에 의해 자금입니다. CO는 호주 대학원 보너스 및 CRC - 입찰 보조 장학금받는 사람입니다.

1. 세포 배양 및 실험 설정 <ol><li> 인간 keratinocytes는 keratinocyte - SFM 15 ML (K - SFM)이있는 75cm 두 조직 문화 flasks에 monolayers로 유지 L - 글루타민 (2 ㎜), 표피 성장 인자 (5 NG와 보충 매체 (GIBCO, 혈청이없는 배지) / ML), 소 뇌하수체 추출 (40 μg / ML) 20 MG / ML gentamicin. 세포 ° C.에게 37 humidified 5 % CO 2 환경에서 재배 </li><li> 전지는 K - SFM 매체의 5 ML을 포함 12 X 25cm 두 조직 문화 flasks에 씨앗을 품고 있습니다. </li></ol> 단세포 suspensions는 trypsinization에 의해 준비가되어 있습니다. 세포는 칼륨이 염분 버퍼와 세탁 및 50~10분에 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 솔루션을 incubated 있습니다. 세포가되기 시작하면 반올림과 ~ 30 %가 떨어뜨려 10 % 태아 소 혈청이 포함된 Dulbecco의 수정 독수리 매체 3 볼륨이 트립신을 중화에 추가됩니다. 세포는 (20 번)와 아래로 pipetting에 의해 떨어뜨려졌다. 세포는 hemocytometer를 사용하여 계산됩니다. 적절한 셀 번호는 세포주 (인간 FEP - 1811 keratinocytes에 대한 약 20 시간)의 두 배로 시간에 따라 씨앗을 품고 있습니다. 목적은 실험 당일 ~ 90 % confluency을 (술병 회 ~ 10 6 세포) 달성하는 것입니다. 12 flasks 구성 실험 약 4 시간에 완료할 수 단일 clonogenic 분석 (6 unirradiated 제어 여섯 조사 flasks)에 최적입니다. 2. 치료 및 조사 <ol><li> 관련 방사선 - 수정 화합물과 적절한 시간에 세포를 치료하고 이온화 방사선 γ - 방사선 또는 X - 선 중 하나에 세포를 노출. </li></ol> 일반적으로 여섯 flasks는 도금 효율 (치료)와 약물에만 컨트롤로 제공하고 있습니다. 다른 여섯 flasks가 조사하고 있습니다. 1 시간, 수용성 천연 항산화 제제,이 예제에서 인간 keratinocytes는 Cinnulin PF의 다양한 농도 (20 μg / ML은 아래와 같습니다에 대한 대표적인 데이터를,, 무결성 Nutraceuticals 국제, 스프링 힐, TN, 미국 CPF)로 처리됩니다 37 ° C. 세포는 137 고사 소스를 (; Nordion 국제, ON, 캐나다, 1.6 쥐 / 분 Gammacell 1000 엘리트 irradiator)를 사용하여 4 쥐와 함께 조사하고 있습니다. 3. 도금 <ol><li> 앞에서 설명한로 치료에 따라, 단일 셀 suspensions을 얻을 수 있습니다. </li><li> 각각의 샘플에있는 세포의 숫자는 신중하게 hemocytometer를 사용하여 계산과 같은 것이 적절한 세포 번호가 (5가 15mm 요리 각각의 복제) 배양 접시에 씨앗을 품고 아르 희석 수 있습니다. 판 당 종자로 세포의 수를 결정할 때 도금 효율 및 / 또는 분수에서 살아남는은 예상해야합니다. 150 식민지 - 목표는 20 ~의 범위를 달성하는 것입니다. </li> 배양 접시는 humidified 플라스틱 복제 상자에서 정렬 및 ° C 콜로니 형성 37 5 % CO 2 환경에서 incubated입니다. </ol> 콜로니 형성을위한 인큐베이션 시간이 서로 다른 세포 라인 1-3 주간 다릅니다, 그것은 시간이 적어도 6 셀 부문에 해당되어야 인정합니다. 이 예제에서는, 인간 keratinocytes에 대한 제어 요리 팔일 50 개 이상의 세포로 이루어진 충분히 많은 클론을 형성해야합니다. 4. 식민지를 해결하고 스테 이닝 펌 후드에서 다음 단계를 완료합니다. <ol><li> 부드럽게 흡인하여 접시의 각 미디어를 제거합니다. </li><li> 5 ML 0.9 %의 식염수에 각각의 접시를 씻으십시오. </li><li> 15-30분 5 ML 10% 중립 버퍼 포르말린 용액으로 식민지를 수정. </li><li> 30~60분에 대한 DH 2 O 5 ML 0.01 % (W / V) 크리스탈 바이올렛과 얼룩. </li><li> DH 2 O와 함께 여분의 크리스탈 바이올렛 씻고 요리 건조 수 있습니다. </li></ol> 5. 콜로니 카운팅 Stereomicroscope <ol><li> 50 개 이상의 개별 세포를 포함하는 식민지가 stereomicroscope를 사용하여 계산됩니다. </li></ol> 이미징 소프트웨어를 사용하여 디지털 이미징과 카운트 <ol><li> 식민지의 디지털 이미지는 카메라를 사용하거나 장치를 스캐닝를 얻을 수있다 </li><li> 식민지은 아래 설명 이미지 분석 소프트웨어 패키지를 사용하여 계산됩니다. </li></ol> ImageJ (피지 버전 1.44a)를 사용하여 셀 카운트 <ol><li> 피지에있는 이미지 파일을 열고, 파일 이동 -&gt; 엽니다. </li><li> &gt; 조정 ...-&gt; 임계값 - 8 비트 형식으로 이미지를 변환 필요한 경우, 이미지로 이동합니다. </li><li> 오직 식민지가 감지되도록 불특정 배경 수준을 줄이기 위해 임계값을 조정합니다. </li><li> 다음을 사용하여 식민지를 카운트 : 프로세스로 이동 -&gt; 이진 -&gt; 맥시멈를 찾습니다. </li></ol> 이 이미지 포맷에 대한 소음의 허용 오차는 0으로 설정할 수 있습니다. 저기 저 반짝 이는 불빛 배경 옵션을 쳤다되었는지 확인하고 모든 세포 식민지가 올바르게 등록되었는지 확인하기 위해 감지된 맥시멈를 미리 봅니다.

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> J Exp Med. 103, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88, 570-574 (1991).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 예제에서는 인간 FEP - 1811 keratinocytes 100 μg / ML CPF에 다양한 농도와 치료를했다, 데이터는 37에서 1 시간 20 μg / ML CPF ° C.에 대해 표시됩니다 다음 치료 세포는 137 고사 소스를 (; Nordion 국제, ON, 캐나다, 1.6 쥐 / 분 Gammacell 1000 엘리트 irradiator)를 사용하여 4 쥐와 함께 조사했다. 컨트롤에 대한 치료 및 약물 치료에만 100 세포는 각 배양 접시에있는 도금되었으며 요리 1000 세포가 조사 샘플 도...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca2+/Mg2+.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH2O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>

clonogenic assay: adherent cells

clonogenic (또는 콜로니 형성) 분석은 이상의 50 년 동안 설립되었습니다, 기술을 설명하는 원래의 논문은 1956 1이 공개되었습니다. 분리 방법을 문서화에서 최초 획기적인 연구 문화 1 X - 선 조사 포유 동물 (헬라) 세포에 대한 최초의 방사선 선량 - 반응 곡선을 생성. 제어 치료 세포와 같은 노출 등 다양한 트리 트먼트를받은 세포 사이에, 기본적으로 clonogenic 분석은 생식 생존의 차이 (50 이상 세포의 식민지를 형성하는 하나의 세포 즉 자손을 만들어 세포의 능력)의 평가를 수 방사선을 이온화하는 다양한 화합물 (예 : 세포 독성 대리인) 또는 그 밖의 경우에는 유전자 조작. 분석은 방사선 생물학에서 가장 널리 인정 기술되어 널리 다른 세포 라인의 방사선 감도를 평가에 사용되고 있습니다. 또한, clonogenic 분석은 일반적으로 방사선 수정 화합물의 효능을 모니터링하고 다른 세포 라인의 콜로니 형성 능력에 세포 독성 에이전트 및 다른 안티 암 치료제의 효과를 결정하는 데 사용됩니다. 자기편 세포 라인을 사용하는 전형적인 clonogenic 서바이벌 실험 식민지를 해결하고 얼룩 세 개의 서로 다른 구성 요소 1) 조직 문화 flasks의 세포 monolayer 치료, 2) 단일 세포 suspensions의 준비 및 배양 접시와 3 세포의 적절한 숫자를 도금)을 포함 관련 잠복기 다음, 이것은 세포 라인에 따라, 1-3 주에서 범위 수 있습니다. 여기서 우리는 영원히 인간 keratinocyte 세포 라인의 사용 (FEP - 1811) 2 자기편 세포 라인 clonogenic 분석을 수행하기위한 일반적인 절차를 보여줍니다. 또한, 우리의 목적은 방사선 세포의 노출 후 도금 효율성과 생존 분수의 계산을 포함 clonogenic의 assays의 일반적인 기능을 설명하고, 천연 항산화 제제의 사용과 방사선 반응의 수정을 예시하는 것입니다.

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

생식 가능성을 평가하기위한 clonogenic 분석의 응용은 이상의 50 년 동안 설립되었습니다. 여기 자기편 세포와 clonogenic 분석을 수행하기위한 일반적인 절차를 보여줍니다.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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Biology

47.8K 조회수.  The Alfred Medical Research and Education Precinct. 생식 가능성을 평가하기위한 clonogenic 분석의 응용은 이상의 50 년 동안 설립되었습니다. 여기 자기편 세포와 clonogenic 분석을 수행하기위한 일반적인 절차를 보여줍니다.

비디오: Clonogenic Assay: Adherent Cells

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 ...

연구

생물학

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

자기편 및 비 점착 성의 포유 동물 세포의 현장 Subcellular 분별 (분리)에서

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

종양 세포의 Vasculogenic 활동을 평가 Matrigel 기반 튜브 형성 분석

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

기본 마우스 Trophoblast 셀 및 Trophoblast의 침략 분석의 절연

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR - 기자의 구성과 microRNA를 사용하여 LightSwitch의 루시페라제 분석 시스템과 인간 microRNAs의 확인 대상이 자기편 세포의 모방

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

갭 접합부 시험에 대한 기능 분석; 인듐 - 주석 산화물에 자기편 세포의 전기 천공 법 (electroporation)

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

동적에 대 한 렌즈 무료 비디오 현미경 및 부착 세포 배양의 정량 분석

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...