El apoyo del Instituto Australiano de Ciencia Nuclear e Ingeniería se reconoce. TCK es el ganador de premios AINSE. Laboratorio de Medicina epigenómico el apoyo de la Nacional de Salud y Consejo de Investigación Médica de Australia (566.559). HR es apoyado por un australiano de post-grado y premios BakerIDI brillante chispa. Este trabajo está financiado por el CRC de Imágenes Biomédicas Development Ltd (CRC-BID), creado y apoyado en cooperación del gobierno australiano de investigación del programa de Centros. CO es el receptor de un australiano de post-grado de premios y una beca complementaria CRC-BID.

1. Cultivo de células y experimental puesta en marcha <ol><li> Queratinocitos humanos se mantienen como monocapas en 75 cm 2 frascos de cultivo de tejidos que contienen 15 ml de queratinocitos-SFM (K-SFM) (GIBCO, medio libre de suero), complementado con L-glutamina (2 mM), factor de crecimiento epidérmico (5 ng / mL), extracto de pituitaria bovina (40 pg / ml) y gentamicina 20 mg / ml. Las células se cultivan en un humidificado 5% de CO2 el medio ambiente a 37 ° C. </li><li> Se siembran las células en 12 x 25 cm 2 frascos de cultivo de tejidos que contienen 5 ml de K-SFM medio. </li></ol> Suspensiones de células individuales son preparadas por tripsinización. Las células se lavan con tampón fosfato salino y se incubaron con un 0,05% de tripsina / EDTA durante 5-10 minutos. Cuando las células empiezan a ser redondeados y aproximadamente el 30% son separados, 3 volúmenes de Dulbecco modificado medio de Eagle con 10% de suero fetal bovino se agrega para neutralizar la tripsina. Las células se separan por pipeteando arriba y abajo (20 veces). Las células se contaron usando un hemocitómetro. Un número adecuado de células son clasificados de acuerdo con el tiempo de duplicación de la línea celular (aproximadamente 20 horas para el consumo humano FEP-1811 queratinocitos). El objetivo es lograr la confluencia de ~ 90% (~ 10 6 células por frasco) en el día del experimento. Un experimento que consta de 12 frascos es óptimo para un solo ensayo clonogénico (seis de control no irradiados y seis frascos irradiados) que puede ser completado en aproximadamente cuatro horas. 2. El tratamiento y la irradiación <ol><li> Tratamiento de las células durante el tiempo necesario con una relevante modificación de la radiación compuesto y exponer las células a las radiaciones ionizantes o γ-radiación o radiografías. </li></ol> Por lo general seis frascos servir como eficiencia de plaqueo (sin tratamiento) y los controles de drogas solamente. Los otros seis frascos son irradiados. En este ejemplo, los queratinocitos humanos son tratados con distintas concentraciones de Cinnulin PF (CPF; Nutracéuticos Internacional de Integridad, Spring Hill, TN, EE.UU., los datos representativos de 20 mg / ml se muestra a continuación), soluble en agua formulación antioxidante natural, durante 1 hora a 37 ° C. Las células son irradiadas con 4 Gy utilizando una fuente de 137 Cs (Gammacell 1000 irradiador Elite; Nordion Internacional, ON, Canadá; 1,6 Gy / min). 3. Enchapado <ol><li> Después del tratamiento, suspensiones de células individuales se obtienen como se describió anteriormente. </li><li> El número de células en cada muestra se contó con cuidado utilizando un hemocitómetro y se diluye de tal manera que el número de células apropiadas se siembran en placas de Petri (cinco réplicas de cada uno en 15 mm platos). Las planchas de eficiencia y / o sobrevivientes fracción debe preverse la hora de decidir el número de células a las semillas por placa. El objetivo es alcanzar un rango de entre 20 a 150 colonias. </li> Placas de Petri están dispuestos en una caja de plástico de la clonación humidificado y se incubaron en un 5% de CO2 el medio ambiente a 37 ° C para la formación de colonias. </ol> El tiempo de incubación para la formación de colonias varía de 1-3 semanas para las líneas celulares diferentes, se acepta que el tiempo debe ser equivalente a por lo menos seis divisiones celulares. En este ejemplo, los platos de control de los queratinocitos humanos requieren ocho días para formar clones lo suficientemente grande que consta de 50 o más células. 4. Fijación y tinción de las colonias Complete los siguientes pasos en una campana extractora. <ol><li> Retire con cuidado los medios de comunicación de cada una de las placas por aspiración. </li><li> Lave cada placa con 5 ml de solución salina al 0,9%. </li><li> Fijar las colonias con 5 ml de 10% neutral solución de formalina tamponada durante 15-30 minutos. </li><li> Mancha con 5 ml 0,01% (w / v) de cristal violeta en dH 2 O durante 30-60 minutos. </li><li> Lavar el exceso de cristal violeta con dH 2 O y deje que los platos se sequen. </li></ol> 5. Recuento de colonias Estereomicroscopio <ol><li> Las colonias que contienen más de 50 células individuales se cuentan utilizando un microscopio estereoscópico. </li></ol> La imagen digital y contar con software de imágenes <ol><li> Las imágenes digitales de las colonias se han obtenido utilizando una cámara o un dispositivo de escaneo </li><li> Se cuentan las colonias utilizando imágenes de los paquetes de software de análisis como se describe a continuación. </li></ol> Conteo de células con ImageJ (Fiji Versión 1.44a) <ol><li> Abra el archivo de imagen en Fiji, vaya a Archivo -&gt; Abrir. </li><li> Si es necesario convertir la imagen en formato de 8 bits, vaya a Imagen -&gt; Ajustar umbral ...-&gt;. </li><li> Ajuste de umbral para reducir los niveles de fondo no específica de manera que sólo las colonias se detectan. </li><li> Contar las colonias con lo siguiente: Ir al proceso - binario&gt; -&gt; Buscar máximos. </li></ol> Para este formato de imagen, la tolerancia al ruido puede ser ajustado a 0. Asegúrese de que la opción de fondo claro está marcada y vista previa de la máxima detectada para comprobar que todas las colonias de células se han registrado correctamente.

<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>

En este ejemplo humano FEP-1811 queratinocitos fueron tratadas con diferentes concentraciones de hasta 100 mg / mL ACB, se muestran los datos de 20 mg / ml ACB durante 1 hora a 37 ° C. Después de celdas de tratamiento fueron irradiados con 4 Gy utilizando una fuente de 137 Cs (Gammacell 1000 irradiador Elite; Nordion Internacional, ON, Canadá; 1,6 Gy / min). Para los tratamientos de control no tratado sólo de drogas y 100 células fueron sembradas en cada placa de Petri y 1000 células por placa se coloca...

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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>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

El clonigénicas (o formadoras de colonias) de ensayo se ha establecido desde hace más de 50 años, el documento original que describe la técnica se publicó en 1956 1. Aparte de la documentación del método, el estudio de referencia inicial generó la primera dosis de radiación curva de respuesta de rayos X irradiados mamíferos (HeLa) las células en cultivo 1. Básicamente, el ensayo clonogénico permite una evaluación de las diferencias en la viabilidad de la reproducción (la capacidad de las células para producir descendencia, es decir, una sola célula para formar una colonia de 50 o más células) entre las células control no tratadas y las células que han sido sometidos a diversos tratamientos, como la exposición a las radiaciones ionizantes, diversos compuestos químicos (por ejemplo, agentes citotóxicos) o en otros casos la manipulación genética. El ensayo se ha convertido en la técnica más ampliamente aceptada en biología de la radiación y ha sido ampliamente utilizada para evaluar la sensibilidad a la radiación de las diferentes líneas celulares. Además, el ensayo clonogénico se utiliza comúnmente para controlar la eficacia de los compuestos de la radiación y la modificación para determinar los efectos de los agentes citotóxicos y otras terapias contra el cáncer en la capacidad de formación de colonias, en diferentes líneas celulares. Un típico experimento clonigénicas supervivencia utilizando líneas de células adherentes consta de tres componentes distintos: 1) el tratamiento de la monocapa celular en frascos de cultivo de tejidos, 2) la preparación de suspensiones de células individuales y las planchas de un número adecuado de células en placas de Petri y 3) la fijación y la tinción de las colonias después de un período de incubación correspondiente, que podría oscilar entre 1-3 semanas, dependiendo de la línea celular. Aquí se demuestra el procedimiento general para llevar a cabo el ensayo clonogénico con líneas de células adherentes con el uso de un inmortalizado línea celular humana de queratinocitos (FEP-1811) 2. Además, nuestros objetivos son describir las características comunes de los ensayos clonogénicos incluyendo el cálculo de la eficiencia de plaqueo y las fracciones de la supervivencia después de la exposición de las células a la radiación, y para ejemplificar la modificación de la radiación de respuesta con el uso de una formulación antioxidante natural.

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

Clonogenic Assay: Adherent Cells

La aplicabilidad del ensayo para evaluar la viabilidad clonigénicas reproductiva se ha establecido desde hace más de 50 años. Aquí se demuestra el procedimiento general para llevar a cabo el ensayo clonogénico con células adherentes.

Ensayo clonigénicas: las células adherentes

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

Research

JoVE Journal

Biology

47.6K vistas.  The Alfred Medical Research and Education Precinct. La aplicabilidad del ensayo para evaluar la viabilidad clonigénicas reproductiva se ha establecido desde hace más de 50 años. Aquí se demuestra el procedimiento general para llevar a cabo el ensayo clonogénico con células adherentes.

Video: 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.

Fabricación y operación de una inserción de oxígeno para Adherente cultivos celulares

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

Investigación

Biología

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.

En Fraccionamiento subcelular situ de células mamíferas adherentes y no adherentes-

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

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.

El aislamiento de células primarias de ratón trofoblasto y ensayo de invasión trofoblasto

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.

Objetivos la identificación de los microARN humanos con el sistema de ensayo con luciferasa LightSwitch reportero 3&#39;UTR-Construye y microRNA un imitador de las células adherentes

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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

Un ensayo funcional para Gap Junctional examen; La electroporación de células adherentes de indio-óxido de estaño

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.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

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.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

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.

Lente libre Video microscopia para la dinámica y el análisis cuantitativo del cultivo de células adherentes

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

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Ensayo de co-inmunoprecipitación utilizando proteínas nucleares endógenas de las células cultivadas bajo condiciones hipóxicas

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...