<ul style="list-style-type:none;"><li> K. Wong, Wong STC son apoyados por el financiamiento del Funcional y Biología Molecular Imaging Center, del Hospital Brigham y de Mujeres </li><li> K. Wong, Wong STC son apoyados por el Centro de Biotecnología e Informática, el Instituto de Investigación del Hospital Metodista, Colegio Médico Weill Cornell. </li><li> KK Kwong, I. Chen, JQ Ren son apoyados por el financiamiento de la Athinoula A. Martino Centro de Imágenes Biomédicas, Hospital General de Massachusetts. </li><li> Lenuta Kloetzer, Braden Kuo son apoyadas con fondos del Centro de Investigación Neuroenteric, Hospital General de Massachusetts </li><li> Los autores agradecen a los Dres. P. Zeng Xu X. y de imágenes ópticas de laboratorio, del Hospital Brigham y de Mujeres por la ayuda técnica. </li></ul>

<ol><li> Mujer HO-1-luciferasa ratones transgénicos (edad de 4-6 semanas) se pueden comprar de Taconic Farms, Inc. A su llegada, los ratones se colocan en un centro de alojamiento de los animales durante al menos 4 días para permitir el alojamiento. </li><li> Preparación para obtener imágenes de bioluminiscencia: <ol style="list-style-type:lower-alpha;"><li> Pese cada ratón HO-1. Peso de los animales es necesaria para calcular la dosis de la luciferina. </li><li> La eliminación del vello: Utilice un algodón para aplicar el removedor de pelo Nair ® sobre el abdomen y / o en la espalda del animal. Espera de 5 a 10 segundos. Utilice un algodón limpio para limpiar el pelo. Sumerja un pedazo de tejido en agua destilada para limpiar el cabello limpio restantes. </li><li> Coloque un papel negro no fluorescente (Strathmore Artagain ® de papel negro) en la plataforma de imágenes de una estación de IVIS 100 para reducir el ruido de fondo. </li><li> Prepare luciferina (de Xenogen Corporation) solución a una concentración de 7,5 mg / ml (disuelto en agua estéril H 2 O). La dosis de luciferina es 65.5 mg / kg de peso corporal. Por lo tanto, un ratón de 20 gramos requiere la inyección intraperitoneal de 0,175 ml de solución de luciferina. Para lograr esto, el volumen de inyección única de solución de luciferina (0.175ml) está precargado con una jeringa con una aguja de calibre 26 </li></ol></li><li> Gua Sha procedimiento: Gua Sha se aplica sólo una vez antes de ejecutar el protocolo de imágenes de bioluminiscencia. Desde Gua Sha no es doloroso, el ratón sólo tiene que ser breve anestesiados por isoflurano a mantener la calma. Gua Sha procedimiento incluye los siguientes pasos: <ol style="list-style-type:lower-alpha;"><li> Aplicar aceite de cocina o agua destilada para lubricar las áreas de piel blanco de Gua Sha un par de veces durante el procedimiento de Gua Sha. </li><li> Repetitiva raspar la zona libre de vello de la espalda del ratón en la fuerza suave pero firme con una cuchara de sopa de cerámica o una cuchara de plástico. </li><li> El raspado continúa hasta que la piel de la espalda se vuelve rojo, que es un signo de la extravasación de sangre por vía subcutánea, alcanza generalmente dentro de 2 a 3 minutos. </li></ol></li><li> Imágenes de bioluminiscencia se puede iniciar inmediatamente o varias horas después de Gua Sha como HO-1 upregulation se construye poco a poco más de un par de horas. El procedimiento para obtener imágenes de bioluminiscencia es la siguiente: <ol style="list-style-type:lower-alpha;"><li> Anestesiar el ratón en una cámara de inducción de la anestesia llena con una mezcla de aire grado isoflurano (1,5%) y médicos. </li><li> Una vez que el ratón está anestesiado, mueva el ratón a la cámara de imágenes en la estación de IVIS 100 imágenes ópticas. Coloque el ratón en una posición supina (abdomen hacia arriba). La cámara de imágenes continuamente infundido con un 1,5% de isoflurano. La plataforma de imágenes se calienta a 37 ° C para mantener el ratón caliente. </li><li> Establecer la adquisición de imágenes en el &quot;binning medio&quot; y el tiempo de ajustar la exposición a 30 segundos. Comienzan a adquirir las imágenes. Configurar el equipo para repetir la adquisición de imágenes cada tres minutos, ya sea manual o automática. Después de la adquisición de la primera imagen, una región de interés (ROI) se dibuja para cubrir el pecho del abdomen, y la zona de la cabeza. Este retorno de la inversión se copia y se pega a las siguientes imágenes con &quot;imagen viva ®&quot;, el software que acompaña a la estación de IVIS 100. </li><li> Intensidad de la señal se mide en forma de fotones por segundo. El tiempo marca la intensidad de la señal para llegar a su valor máximo y mantener en la imagen del ratón durante unos 5 a 10 minutos después de la hora pico. </li><li> Cuando la adquisición de imágenes en la posición supina se hace, dar la vuelta el ratón por encima y ponerla en la posición prona (copia de seguridad). Continuar imagen del animal de 5 a 10 minutos con el retorno de la inversión ya elaborado para cubrir la espalda y la zona de la cabeza en el uso de la &quot;imagen viva ®&quot;. La elección de la imagen de la posición boca abajo después de la posición supina es arbitraria. Se puede optar por comenzar con la posición boca abajo, en función de los órganos principales de interés. Otra opción es la imagen de la posición decúbito supino y prono en experimentos separados. </li><li> Cuando la adquisición de imágenes en la posición prona se termine, apague el isoflurano y el movimiento del ratón de la cámara de imágenes de la cámara de inducción para recuperarse. La cámara de inducción está impregnada de aire medicinal y sólo el ratón por lo general se despierta en menos de un minuto. </li><li> Guardar datos de imágenes para el procesamiento posterior. </li><li> Tras el estudio de imagen inicial, adquisiciones adicionales de imágenes se llevan a cabo durante varios días después de Gua Sha. Un programa de muestreo de imagen se repite en la 12 ª hora, 24 ª hora, 36 ª hora, 48 ª hora, 72 horas ª y 120 ª hora, pero el momento exacto de la programación de imágenes es flexible. Sesiones adicionales de imágenes se pueden agregar si un seguimiento más largo es necesario. Antes de imágenes, repita el procedimiento de eliminación de pelo si el pelo se ha vuelto a crecer. </li></ol></li><li> Para el grupo control, repetir todo el procedimiento, pero vaya Gua Sha (paso 3). Si el mismo grupo de ratones se utiliza para el control y Gua Sha, la exp de controleriment debe llevarse a cabo preferentemente antes del procedimiento Gua Sha o por lo menos un mes después. </li></ol> Los resultados representativos: Las imágenes de bioluminiscencia en la figura 1 muestra en vivo de la regulación al alza de HO-1 en respuesta a Gua Sha. El gráfico de la figura 2 se muestra la evolución cuantitativa temporal de más de 120 horas de flujo óptico (fotones / seg) de todo el cuerpo del ratón es el mismo en relación con Gua Sha ... <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg"><img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="la figura 1" border="0" /></a> Figura 1. De izquierda a derecha las imágenes, representante de la vista frontal (decúbito supino) del mismo ratón antes de Gua Sha, a las 18 horas, 36 horas y 120 horas después de Gua Sha, respectivamente. Después de Gua Sha, se observa el progreso del aumento significativo de la intensidad de la señal en múltiples órganos que abarcan las regiones del tracto gastrointestinal, el tracto genital, el hígado, los riñones (desde la vista atrás, no se muestra), y otros. <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg"><img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="la figura 2" border="0" /></a> Figura 2. Cambio cuantitativo de flujo (fotones / seg) de todo el cuerpo un seguimiento de más de 120 horas después de Gua Sha en el mismo ratón de la figura 1.

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The authors have nothing to disclose.

Transcripción de la HO-1, una forma inducible de la hemo oxigenasa, es aumentada por muchos factores, incluyendo hemo, el peróxido de hidrógeno, la radiación UV, la hipoxia y el estrés físico. Imágenes de cuerpo entero, como el reportado HO-1 protocolo de bioluminiscencia proporciona un panorama general de la sistémica HO-1 de expresión en múltiples órganos. En pequeños animales, imágenes de bioluminiscencia que permite una alta sensibilidad in vivo cuantitativa en tiempo real la evaluación longitudinal ó...

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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

Gua Sha es una terapia tradicional popular chino que emplea raspado de la piel a causa subcutánea extravasación microvascular y contusiones. El protocolo para obtener imágenes de bioluminiscencia óptico de HO-1-<em&gt; Luciferasa</emRatones&gt; transgénico reportados en este manuscrito ofrece una rápida<em&gt; In vivo</emAnálisis&gt; de la regulación al alza de la heme oxigenasa-1 (HO-1) la expresión génica en respuesta al procedimiento Gua Sha. HO-1 ha sido durante mucho tiempo se sabe que proporcionan citoprotección contra el estrés oxidativo. La regulación al alza de la HO-1, evaluada por la salida de la bioluminiscencia, se cree que representan una respuesta antioxidante de los productos de hemoglobina que circulan publicado por Gua Sha. Gua Sha fue administrado por los golpes repetidos de una cuchara de borde suave sobre la piel lubricada en la espalda u otra parte del cuerpo blanco del ratón transgénico hasta petequias (hemorragias en astillas) o equimosis (moretones) indicativo de la extravasación de sangre de los capilares subcutáneos se observó. Después de Gua Sha, las sesiones de la bioluminiscencia de imágenes se llevó a cabo al día durante varios días para seguir la dinámica de la HO-1 de expresión en múltiples órganos internos.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha, tradicional raspado de piel chino terapéutico, las causas subcutánea extravasación de sangre microvascular. Se presenta un protocolo de imágenes de bioluminiscencia de HO-1-<em&gt; Luciferasa</em&gt; Ratones transgénicos para demostrar que Gua Sha upregulates hemo oxigenasa-1 (HO-1) en múltiples órganos.

Bioluminiscencia de imágenes de hemo oxigenasa-1 Upregulation en el procedimiento de Gua Sha

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

13.9K vistas.  Massachusetts General Hospital, Harvard Medical School. Gua Sha, tradicional raspado de piel chino terapéutico, las causas subcutánea extravasación de sangre microvascular. Se presenta un protocolo de imágenes de bioluminiscencia de HO-1- Luciferasa Ratones transgénicos para demostrar que Gua Sha upregulates hemo oxigenasa-1 (HO-1) en múltiples órganos.

Video: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Un procedimiento de cirugía craneotomía para imágenes cerebrales crónicas

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Investigación

Biología

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro e in vivo de imágenes de bioluminiscencia gen reportero de células estaminales embrionarias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Procedimiento murino Trasplante Renal

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagen funcional de grasa marrón en ratones con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Monitoreo de células autónomas Ritmos reloj circadiano de expresión génica utilizando luciferase reporteros Bioluminiscencia

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Obtención de imágenes de peróxido mitocondrial en células de levadura vivas mediante mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Autor destacado: Avance de la investigación mitocondrial - Biosensor mtHyper7 para análisis subcelular 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Obtención de imágenes de mtHyPer7, un biosensor radiométrico para el peróxido mitocondrial, en células vivas de levadura

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...