Name: techniques to induce and quantify cellular senescence
Uploaded: 2017-05-01T11:30:00
Description: Watch this Scientific Journal Video about Techniques to Induce and Quantify Cellular Senescence at JoVE.com

Это исследование было поддержано исследовательской программой Intramural Национальных институтов здравоохранения, Национальный институт по проблемам старения. Авторы хотели бы поблагодарить Мириам Горосп и Котб Абдельмохсен за полезные дискуссии о старении и Котб Абдельмохсна для также критически чтения рукописи. Мы также благодарим наших лабораторных членов, особенно Дуглас Длузен для критически чтении рукописи.

1. побуждающих Репликативного Старение <ol><li> Оттаивания низкого прохода фибробласты диплоидного человека (например, WI-38 и IMR-90) или другие клеточные линии. Примечание: В данном случае были использованы диплоидные фибробласты человека, но эти протоколы могут быть использованы д...

Здесь мы описали способы репликативной и ДНК-повреждений, вызванных старением с использованием диплоидных фибробластов человека. Кроме того, методы для количественного определения белка и уровни мРНК различных стареющих-ассоциированных белков включены, а также для окрашивания SA-бет...

Более полувека назад, Хейфлик и его коллеги описали , как нетрансформированный клетки пролиферируют в культуре, но только в течение ограниченного периода времени 1. Долгосрочное культивирование человеческих фибробластов вызвало клетку, чтобы остановить пролиферирующие; однако, они были метаболически активными, и это называлось клеточное старение. Старение может быть полезным для ингибирования онкогенеза, но она также может быть вредно, так как это , как полагают, способствуют потере регенерационной способности , что происходит со старением 2, 3. Стареющие клетки , как были показаны, накапливается в тканях , как люди возраст 4 и участвуют в ряде биологических процессов, в том числе эмбрионального развития, заживления ран, восстановления тканей, а также связанные с возрастом воспаления 2. Постоянные пассажи клеток в культуре индуцирует репликативное старение, который был связанна теломер истощение и нестабильность генома. Различные напряжения клеток, в том числе повреждения ДНК и онкогенов, могут также вызвать старение 3. Старение вызвано другими , чем теломер истощение факторов часто называют стрессом-индуцированным или преждевременным старением и , как правило , зависит от р16 Ink4a / Rb пути 5. Несмотря на то, пролиферирующих, нетрансформированные клетки обычно появляются шпиндель в форме, стареющие клетки могут быть идентифицированы как имеющие определенные характеристики, в том числе плоскую, большую морфологию и повышенную старении-ассоциированной β-галактозидазы (SA-β-гал) (фиг.1 и 2). Стареющие клетки также накапливаются маркеры повреждения ДНК, в том числе гамма-H2AX (рисунок 3) 6, и, потенциально, Старение-ассоциированные гетерохроматина очаги (SAHF) (Рисунок 4) 7. Стареющие клетки имеют более высокие уровни регуляторов клеточного цикла, в том числе р<html> 16 (р16 INK4A) и / или р21 и р53 (рисунок 5) 8, 9. Кроме того, недавние данные показали , что стареющие клетки могут иметь неавтономных эффекты, секретирующие ряд про-воспалительных цитокинов и хемокинов называется Старение-ассоциированный секреторной фенотип (SASP) 10. Хотя это явление SASP может варьироваться в зависимости от типа клеток типа клеток, в общем, это свидетельствует увеличение интерлейкина-6 (ИЛ-6), ИЛ-8, гранулоцитарно-макрофагальный колониестимулирующий фактор (GM-CSF), growth- регулируемый онкогена α (GRO-α), а также GRO-β, среди других (рисунок 6). Частности , стресс или повреждение , которое вызывает старение может также влиять на секреторную фенотип 11, 12, 13. SASP может быть обнаружен путем измерения уровней секретируемых белков с помощью ELISA, или цитокина / белковых массивовs = &quot;Xref&quot;&gt; 10, 14. Хотя пост-транскрипционные механизмы могут регулировать уровни белка SASP 11, 15, 16, 17, изменения в уровне мРНК также могут быть обнаружены во многих случаях. Эти изменения, как правило, более чувствительны и проще, чем для количественного измерения уровня белка. Другие стареющие маркеры также могут быть оценены, включая стойкие повреждения ДНК ядерных очаги, называемые сегменты ДНК с хроматином изменениями армирующих старения (ДНК-Рубцы) 18, а также различных другими маркерами 3, 19, 20. Здесь мы опишем общие методы для индукции старении в клетках в культуре, а также для измерения нескольких маркеров старения, включая SA-бета-Gal, гамма-H2AX, SAHF, и белок и мРНК стареютсть-ассоциированных молекул.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Tris-glycine gels</td>
			<td>Invitrogen</td>
			<td colspan="2">XP00160BOX</td>
		</tr>
		<tr>
			<td>Acid-Phenol ChCl3</td>
			<td>Ambion</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-Fluor 568 goat anti-mouse antibody</td>
			<td>Invitrogen</td>
			<td>A11031</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Cell lifters</td>
			<td>Corning Inc.</td>
			<td>3008</td>
			<td>Cell scraper</td>
		</tr>
		<tr>
			<td>ECL anti-mouse HRP linked antibody</td>
			<td>Amersham</td>
			<td>NA931V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Plus Western Blotting Substrate</td>
			<td>Pierce</td>
			<td>32132</td>
			<td>ECL</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>MP01306</td>
			<td>stock 5 mg/ml in dH2O</td>
		</tr>
		<tr>
			<td>GAPDH antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32233</td>
			<td>1:1,000-5,000 dilution</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone H3 dimethyl K9 monoclonal antibody</td>
			<td>Abcam</td>
			<td>1220</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Human IL-6 Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>D6050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>D8000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GROa Quantikine ELISA assay</td>
			<td>R&#38;D systems</td>
			<td>DRG00</td>
			<td></td>
		</tr>
		<tr>
			<td>N-N-dimethylformamide&#160;</td>
			<td>Sigma</td>
			<td>D4551</td>
			<td>DMF</td>
		</tr>
		<tr>
			<td>p16 monoclonal antibody</td>
			<td>BD Biosciences</td>
			<td>51-1325gr</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>p21 monoclonal antibody</td>
			<td>Millipore</td>
			<td>05-345</td>
			<td>1:750 dilution</td>
		</tr>
		<tr>
			<td>p53 monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td>sc-126</td>
			<td>1:500 dilution clone DO-1</td>
		</tr>
		<tr>
			<td>phospho-H2AX (Ser139) FITC conjugate antibody</td>
			<td>Cell Signaling</td>
			<td>9719</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>POWER SYBR-green PCR master mix&#160;</td>
			<td>Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-stained molecular weight markers</td>
			<td>Biorad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade&#160;</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane&#160;</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Senescence b-Galactosidase Staining Kit</td>
			<td>Cell Signaling</td>
			<td>9860</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Ambion/Life Tech</td>
			<td>10296028</td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques to induce and quantify cellular senescence

В ответ на клеточный стресс или повреждения, пролиферирующие клетки могут вызвать определенную программу, которая инициирует состояние долгосрочной остановки клеточного цикла, называемую клеточным старение. Накопление стареющих клеток происходит с организменном старения и за счет непрерывного культивирования в пробирке. Стареющие клетки влияют на многие биологические процессы, в том числе эмбрионального развития, ремонта и регенерации тканей, подавление опухоли, и старение. Клейма стареющие клетки включают в себя, но не ограничиваются ими, увеличилась Старение-ассоциированные β-галактозидазы (SA-бета-гал); р16 Ink4a, p53, p21 и уровней; более высокие уровни повреждений ДНК, в том числе гамма-H2AX; формирование Старение-ассоциированного гетерохроматина очагах (SAHF); и приобретение старении-ассоциированной секреторного фенотипа (SASP), явление, характеризующееся секреции ряда провоспалительных цитокинов и сигнальных молекул. Здесь мы описываем протоколы для обоих репликативной иПовреждения ДНК-индуцированное старение в культивируемых клетках. Кроме того, мы выделим методу для мониторинга стареющего фенотипа с использованием несколько старения-ассоциированными маркеров, включая SA-бету-гала, гамма-H2AX и SAHF окрашивание и количественную оценку уровней белка и мРНК регуляторов клеточного цикла и SASP факторов. Эти методы могут быть применены к оценке старения в различных моделях и тканях.

На рисунках 2 - 6 показывают репрезентативные результаты от SA-β-гал окрашивания; окрашивания для гамма-H2AX и SAHF; оценка уровней белка р16, р21 Ink4a и р53; и мРНК и уровни белка стареющих-ассоциированных молекул. Увеличение SA-β-гал окрашивание происходит с реплик?...

Watch this Scientific Journal Video about Techniques to Induce and Quantify Cellular Senescence at JoVE.com

Techniques to Induce and Quantify Cellular Senescence

Клеточное старение, необратимое состояние остановки клеточного цикла, может быть вызвано различными клеточными напряжениями. Здесь мы описываем протоколы для индукции клеточного старения и методов оценки маркеров старения.

Методы индуцируют и Quantify Cellular Старение

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

The overall goal of these techniques is to provide a toolbox for enabling the induction, monitoring, and quantification of cellular senescence. These methods can help answer key questions about the identification and quantification of cellular senescence and the biologic factors that regulate cellular and organismal aging. The main advantage of these techniques is that they encompass a variety of methods for monitoring cellular senescence in different models, tissues, and cell types.Begin by plating the early passage proliferative cells of interest at a sparse density in the appropriate dish for the analysis. And culture the cells overnight in a tissue culture incubator. When the cells are 50-60%confluent, aspirate the growth medium and wash the cells with PBS two times.After the second wash, cover the cells with a thin layer of PBS. And expose the cells to a single 10 grade dose of gamma irradiation. Replace the PBS with growth medium.And return the cells to the incubator. Changing the medium every 48 to 72 hours with daily monitoring for morphological changes under a light microscope. Seven to 10 days after the irradiation, thaw all of the reagents from a commercial senescence associated beta-galactosidase assay kit in a 37 degree Celsius water bath.When all of the reagents reach room temperature, prepare the fixative and staining solutions according to the manufacturer's instructions. And gently wash each well with room temperature PBS. Take care to rinse the cells carefully as senescence cells do not adhere well and can be easily removed during washes.Fix the cells in each well with one milliliter of fixative solution per well for 10 to 15 minutes at room temperature. Followed by two two milliliter washes with PBS per well. Next, add one milliliter of beta-galactosidase staining solution to each well and cover the plate with aluminum foil for a 24 to 48 hour culture in a 37 degrees Celsius incubator without carbon dioxide.Check the cells at 24 hours. When the cells are sufficiently stained, replace the beta-gal solution with fresh PBS and examine the cultures on a light microscope with an attached camera at a 10x or a higher objective. The senescence associated beta-galactosidase cells will appear blue.Then, obtain at least five non-overlapping images per well. And manually count the total number of senescence associated beta-galactosidase positive cells and the total number of cells per image to calculate the percentage of senescence associated beta-galactosidase cells per well. 10 days after gamma irradiation, wash the cells three times with PBS.After the last wash, replace the PBS with 2.5 milliliters of serum free medium, supplemented with antibiotics per six centimeter dish and return the cells to the tissue culture incubator overnight. The next morning, transfer the medium into a conical tube on ice. And wash the cells two times with fresh PBS.Then, trypsinise and count the cells. Next, centrifuge the supernatant harvested from the cell culture plate. And then filter it through a 0.45 micrometer syringe strainer and freeze the medium in 500 microliter aliquots at 80 degree Celsius until their analysis by ELISA.Increased senescence associated beta-gal staining occurs with replicative and DNA damage induced senescence. Note the enlarged flat shape of the senescent cells compared to the spindle-like appearance of the proliferating fibroblasts. In this experiment, proliferating young or replicative senescent cells were fixed as described in the written protocol.And stained with an anti-body against a bio marker for DNA double strand breaks and DAPI. With an apparent increase in the number of DNA double strand break fosi observed in the senescent cells. RT-qPCR analysis of senescence associated secretory phenotype factors reveals that at least some factors are up regulated after gamma irradiation induced senescence in low passage human diploid fibroblasts at the MRNA level.Which is further confirmed by ELISA of the gamma irradiated cell culture supernatants. Once mastered, the DNA damage induction and SA beta-gal staining can be completed in approximately 30 minutes of hands-on time. The condition media for quantifying SASP protein levels can be prepared in about 30 minutes.While attempting this procedure, it's important to remember to monitor them undergoing morphological changes in the cell cultures before proceeding to the senescent marker quantification. After its development, these techniques paved the way for researchers in the fields of aging and cancer to explore senescence in a variety of tissues, cell cultures, and model organisms. After watching this video, you should have a good understanding of how to induce, monitor, and quantify cellular senescence.

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BioMEMS and Cellular Biology: Perspectives and Applications

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology   essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate   has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.

BioMEMS и клеточной биологии: перспективы и приложения

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Микроинъекция методы для изучения митоза в Дрозофилы Синцитиальный эмбрионов

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Несоответствие изображений Ремонт и клеточных реакций на повреждение ДНК в Сенная Bacillus</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi скрининга для выявления Постэмбриональное фенотипы в С. Элеганс</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Длиной теломер и теломеразы, Инь и Ян клеточного старения

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

Простое и эффективное управление и визуализация микрочастиц в системе кровообращения малых рыб с помощью инъекций почек

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Выступ силовой микроскопии: Метод количественного определения сил, разработанный ячейки выступов

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

Носовые разности потенциалов для количественного определения транс эпителиальных ионный транспорт в мышей

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Приложения пространственно временных карт и методы анализа частиц для количественного определения внутриклеточных Ca2 + сигнализации в Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats Using the Thymidine Analog BrdU

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2&#39;-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.

This paper presents four of the most common techniques for visualizing BrdU positive cells to measure adult neurogenesis in rats. This work includes instructions for reagent preparation, thymidine analog administration, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, immunofluorescence, signal amplification, counterstaining, microscopy imaging, and cell analysis.