Il lavoro descritto in questo manoscritto è stato reso possibile da un finanziamento della Fondazione Patrick F. Taylor.

1. Cromosoma raccolta di cellule aderenti <ol><li> Protocollo standard <ol><li> Crescere le cellule in base alle specifiche condizioni di coltura delle cellule. Quando le cellule hanno raggiunto la fase logaritmica (80% di confluenza), aggiungere 10 microlitri / ml di Colcemid al pallone di coltura cellulare. Si raccomanda un minimo di 2 x 10 6 cellule. </li><li> Incubare le cellule a 37 ° C in un incubatore CO 2 5% per 45 min. Usando una pipetta sterile, trasferire file multimediali da cellule in una provetta da 15 ml. Mettere da parte. </li><li> Lavare delicatamente le cellule aggiungendo 2 ml di HBSS tampone nel pallone. Tampone Swirl e poi rimuovere con una pipetta. Scartare. </li><li> Aggiungere 1 ml di tripsina, assicurando che copre l&#39;intera superficie del pallone. Lasciare solo le cellule in tripsina per circa 2 min. Una volta che la maggior parte delle cellule sono distaccato, pipettare i media nel tubo conico indietro sulle cellule. </li><li> Trasferire la sospensione cellulare in aliquote da 10 ml in provette da 15 ml coniche.Centrifugare a 200 xg per 10 min. Rimuovere il surnatante e risospendere il pellet. </li><li> Aggiungere 10 ml di 0,075 M KCl preriscaldata a 37 ° C al pellet rimasto nel tubo conico. Tubo Vortex a velocità media per mescolare KCl e le cellule. </li><li> Incubare le cellule a 37 ° C per 10 min. Centrifugare a 200 xg per 5 min a 25 ° C. Rimuovere il surnatante (fino a circa 0,5 ml resti) e risospendere pellet. </li><li> Aggiungere con cautela 5 ml di fissativo fresco di Carnoy (3:1 rapporto di metanolo: acido acetico glaciale) alle cellule, mentre vortex. Aggiungere quindi 5 ml più di fissativo, senza vortex per un totale di 10 ml. </li><li> Centrifugare a 200 xg per 5 min. Rimuovere le cellule surnatante e sospendere nuovamente. Aggiungere 5 ml di fissativo ad ogni provetta. </li><li> Centrifugare a 200 xg per 5 min. Rimuovere le cellule surnatante e sospendere nuovamente. Aggiungere 5 ml di fissativo ad ogni provetta. Le cellule possono essere conservate a 4 ° C fino ad un anno. </li></ol></li><li> Modifica del protocollo: harvesti cromosoming di linee cellulari linfoblastoidi <ol><li> Crescere le cellule in base alle specifiche condizioni di coltura delle cellule. (Usa pallone appropriato per la quantità di cellule in coltura). Quando le cellule hanno raggiunto la fase logaritmica (circa 2 giorni dopo le cellule sono split), aggiungere 10 microlitri / ml di Colcemid al pallone di coltura cellulare. </li></ol></li><li> Modifica del protocollo: Cromosoma raccolta da sangue intero Fitoemoagglutinina (PHA), una lectina derivato dal fagiolo rosso, è un potente mitogeno per le cellule T umane 16. 72 ore dopo l&#39;aggiunta di PHA alla cultura, circa il 45% delle cellule sono in fase S. Questo rappresenta il picco di attività mitotica, ed è il punto ottimale in cui per raccogliere per gli studi cromosomici. Altri mitogeni come pokeweed (1-10 mg / ml) possono essere utilizzati nell&#39;analisi cellule B 17,18. <ol><li> Raccogliere almeno 1 ml di sangue intero in una parte superiore del tubo di sodio eparina verde. Usare entro 3 giorni dopo la raccolta. Sangue Conservare a temperatura ambiente delle Nazioni Unitetil pronto all&#39;uso. </li><li> Aliquotare 0,25 ml di sangue intero in 10 ml di mezzi RPMI completo contenente L-glutammina (siero fetale bovino 20%, 1% di penicillina / streptomicina, 1% fungizone, e 1% PHA). Coltura a 37 ° C con 5% di CO 2. </li></ol></li><li> Modifica del protocollo: Cromosoma raccolta dal midollo osseo Analisi citogenetica sul midollo osseo è utile in molti disturbi ematologici maligni come l&#39;osservazione di un clone cromosomicamente anormale può essere diagnostica per un tipo specifico di leucemia. Studi cromosomiche sono utilizzati anche per valutare la progressione della malattia, come l&#39;insorgenza di crisi blastica e risposta al trattamento. Poiché le cellule del midollo osseo sono attivamente dividono, nessuno stimolo mitogeno è necessaria 9,19. Per le leucemie acute 24 e 48 hr culture sono anche impostati. <ol><li> Raccogliere almeno 1 ml di midollo osseo in una parte superiore del tubo di sodio eparina verde. Aliquotare 0,25 ml di midollo osseo in 10 ml di mezzi RPMI completo contenente L-glutammina (20% di siero bovino fetale, 1% di penicillina / streptomicina, 1% fungizone). Coltura a 37 ° C con 5% di CO 2. </li></ol></li></ol> 2. Presentazione Preparazione e colorazione Solid Una rapida valutazione di una diapositiva rappresentante fornirà informazioni sulla qualità del raccolto prima di procedere ad ulteriori procedure come G-banding, FISH, CGH, o SKY. Alcuni laboratori preferiscono macchia solida alle cellule per la raccolta rapida e la valutazione dei cromosomi. In alternativa, un microscopio a contrasto di fase può essere utilizzata per questa analisi. I vetrini sono meglio preparati quando l&#39;umidità è circa il 50% e la temperatura ambiente (20-25 ° C). <ol><li> Centrifugare le cellule a 200 xg per 5 min a 25 ° C. Rimuovere il surnatante fino a soli 0,3-0,5 ml resti. </li><li> Dopo aver risospeso delicatamente i pellet, pipette tre gocce della sospensione di cellule da una distanza di circa 2 in su un vetrino che è inclinato di un angolo di circa 45 ° e consentire la sospensionerotolare attraverso la diapositiva. Aggiungere una grossa goccia di fissativo fresco di Carnoy alla diapositiva. </li><li> Asciugare il retro del vetrino su un tovagliolo di carta e poi sedersi il vetrino ad asciugare per almeno 10 min. La slitta deve essere completamente asciutto. </li><li> Preparare fresco Giemsa soluzione (rapporto 3:1 di Gurr Buffer e Giemsa). Porre i vetrini in un rack per colorazione. Coprire l&#39;intera diapositiva nella soluzione colorante Giemsa. Lasciare che i vetrini rimangono nella soluzione colorante per 5 min. Sciacquare i vetrini con acqua distillata, drenare, e lasciare asciugare. </li><li> Aggiungere 4 gocce di Permount e un vetrino alla diapositiva. Assicurarsi che non vi siano bolle sotto il vetrino. L&#39;eccesso Permount può essere rimosso con un tovagliolo di carta. </li><li> Analizzare le cellule con un microscopio ottico sotto 10X e 100X ingrandimento. Se le cellule in metafase sono abbondanti e ben diffusione, le diapositive rimanenti possono essere utilizzati per altre procedure sperimentali. </li></ol> 3. G-Banding Uso tripsina e Giemsa (GTG) &lt;p&gt; La tripsina è un enzima proteolitico che denatura istoni eucromatiche nelle regioni del DNA con l&#39;attività trascrizionale maggiore risultante. A seguito di Giemsa, queste regioni appariranno come bande luminose. Cromatina altamente condensato con poca o nessuna attività trascrizionale (eterocromatina) avrà un gran parte delle sue istoni protette dalla tripsina e pertanto macchia scura seguente Giemsa. È essenziale inizialmente G-band una diapositiva a monitorare le condizioni e regolare la temporizzazione tripsina se necessario per le diapositive successive. <ol><li> Aggiungere le seguenti soluzioni a 4 vaschette Coplin. Jar # 1 = 30 ml di 1x HBSS e 4 ml di tripsina 10x (0,5%) Jar # 2 = 50 ml di 1x HBSS Jar ° 3 = 45 ml di 1x HBSS e 5 ml di siero fetale bovino Jar # 4 = 50 ml di 1x HBSS </li><li> Immergere ogni diapositiva in Jar # 1 per 5 secondi, rapido risciacquo in Jar # 2, lasciare ogni diapositiva in Jar # 3 per almeno 30 secondi, rapido risciacquo in Jar # 4 poi permettono vetrini si asciughino. </li><li> Preparare fresh Giemsa colorazione Solution (rapporto 3:1 di Gurr Buffer e Giemsa). Porre i vetrini in un rack per colorazione. Coprire l&#39;intera diapositiva nella soluzione colorante Giemsa. Lasciare che i vetrini rimangono nella soluzione colorante per 5 min. </li><li> Sciacquare i vetrini in acqua distillata nello stesso ordine in cui erano macchiate. Lasciare i vetrini ad asciugare per circa 10 min. La slitta deve essere completamente asciutto. </li><li> Aggiungere 4 gocce di Permount e un vetrino alla diapositiva. Assicurarsi che non vi siano bolle sotto il vetrino. L&#39;eccesso Permount può essere rimosso con un tovagliolo di carta. </li><li> Analizzare le cellule con un microscopio ottico sotto 10X e 100X ingrandimento. Regolare la temporizzazione tripsina del resto delle diapositive sulla base dei risultati della prima slitta. </li></ol>

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Abbiamo utilizzato il presente procedimento per il cromosoma isolamento da cellule di vari organismi, tra cui vari tipi di cellule ottenute da umano, macachi Rhesus, ratti e topi 11,20-22. Il protocollo standard è disponibile, ma alcuni passaggi chiave e variabili potrebbe essere necessario regolare per questi diversi tipi di cellule. Diversi passaggi specifici sono cruciali sia nella preparazione cromosomica e G-bande per garantire la migliore qualità dei risultati. Una delle variabili che possono influenz...

Analisi cromosomica è una tecnica tradizionale utilizzata in tutto il mondo per diagnosticare instabilità cromosomica e riarrangiamenti che portano a malattie genetiche e tumori maligni 1,2,8,9. Inoltre, una risoluzione maggiore per la diagnosi e la ricerca di anomalie genetiche e costituzionali cancro acquisita può essere realizzato con la combinazione delle procedure citogenetiche classiche e metodologie citogenetiche molecolari come ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH) , e cariotipo spettrale (SKY) 14,15. Più recentemente, queste tecniche sono state utilizzate per la valutazione di instabilità cromosomica associata ricerca sulle cellule staminali. Anomalie del cariotipo come aneuploidia di cellule in coltura a lungo termine embrionali (ES) e cellule staminali di diversi organismi sono stati riportati da diversi laboratori. Recenti evidenze sostiene che alcune linee cellulari sono intrinsecamente più inclini al cromosoma instabilità indipendentemente dalle condizioni di coltura. Per questo motivo, al momento di stabilire e / o il mantenimento di linee di cellule staminali umane Rhesus, mouse, o, analisi cromosomica è raccomandata come parte del processo di controllo di qualità. Molti rapporti descrivono il crescente interesse per l&#39;uso di routine e citogenetica molecolare per monitorare la stabilità cromosomica di cellule staminali e cellule maligne o vari organismi in coltura 10-13. Questi protocolli sono sempre più utilizzati dai laboratori non genetici per una rapida valutazione cromosomica delle loro cellule in coltura 13. Presentiamo nostre procedure di base per la preparazione cromosoma da vari tipi di cellule, che possono essere applicate per entrambi gli scopi e le cellule clinici e di ricerca derivati ​​da vari organismi.

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

Cromosoma (citogenetica) analisi è ampiamente utilizzato per il rilevamento di instabilità cromosomica. Quando seguito da G-banding e le tecniche molecolari come ibridazione in situ fluorescente (FISH), questa analisi ha la potente capacità di analizzare le singole celle per le aberrazioni che coinvolgono gli utili o le perdite di porzioni del genoma e riarrangiamenti che coinvolgono uno o più cromosomi. Negli esseri umani, anomalie cromosomiche si verificano in circa 1 ogni 160 nati vivi 1,2, il 60-80% di tutti gli aborti spontanei 3,4, il 10% dei nati morti 2,5, il 13% degli individui con malattia cardiaca congenita 6, 3-6% dei casi di infertilità 2, e in molti pazienti con ritardo dello sviluppo e difetti di nascita 7. Analisi citogenetica di malignità è abitualmente utilizzato dai ricercatori e clinici, come osservazioni di anomalie cromosomiche clonali hanno dimostrato di avere sia un significato diagnostico e prognostico 8,9.60; isolamento Cromosoma è prezioso per la terapia genica e la ricerca sulle cellule staminali degli organismi inclusi primati non umani e roditori 10-13. I cromosomi possono essere isolate da cellule di tessuti vivi, compresi i linfociti del sangue, fibroblasti cutanei, amniociti, placenta, midollo osseo, e campioni tumorali. I cromosomi sono analizzati nella fase metafase della mitosi, quando sono più condensati e quindi più visibile. Il primo passo della tecnica di isolamento cromosoma comporta la rottura delle fibre del fuso mediante incubazione con Colcemid, per evitare che le cellule di procedere alla fase successiva anafase. Le cellule sono poi trattate con una soluzione ipotonica e conservati nel loro stato gonfio di fissativo di Carnoy. Le cellule vengono poi trascinati nella vetrini e possono quindi essere utilizzati per una varietà di procedure. G-banding comprende il trattamento tripsina seguita da colorazione con Giemsa per creare luce caratteristico e bande scure. Lo stesso procedure isolare cromosomi può essere utilizzato per la preparazione di cellule per procedure quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY) 14,15.

Spread metafase alta qualità sono essenziali per l&#39;analisi cromosomica. Un test riuscito produce cromosomi che sono ben diffuse e di adeguata morfologia cromosoma. Cromosomi Correttamente G-banda contengono la luce caratteristico e modelli di bande scure. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> Figura 1. Una diffusione cromosomico suc...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

I cromosomi possono essere isolate da cellule vive come linfociti o fibroblasti cutanei, e da organismi compresi gli esseri umani o topi. Queste preparazioni cromosomiche possono essere ulteriormente utilizzati per routine di G-banding e le procedure di citogenetica molecolare quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY).

Cromosoma Preparazione da cellule in coltura

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.5K Views.  Louisiana State University Health Science Center.  I cromosomi possono essere isolate da cellule vive come linfociti o fibroblasti cutanei, e da organismi compresi gli esseri umani o topi. Queste preparazioni cromosomiche possono essere ulteriormente utilizzati per routine di G-banding e le procedure di citogenetica molecolare quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY). 

Video: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Conteggio e determinazione della vitalità delle cellule in coltura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Ricerca

Biologia

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparazione dei cromosomi di Drosophila Zucche politene per l&#39;etichettatura di anticorpi

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Saggi Reporter massicciamente parallelo in cellule di mammifero in coltura

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e analisi funzionale dei mitocondri da cellule in coltura e Tissue mouse

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Un rapido aria secca cadente Cromosoma Metodo di preparazione adatto per i pesci nelle piante

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparazione del cromosoma meiotico si diffonde da Mouse spermatociti

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

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.

Analisi di co-immunoprecipitazione utilizzando proteine endogene nucleari da cellule coltivate in condizioni di ipossia

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

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Preparazione di cellule progenitrici adipose dai tessuti adiposi epididimali del topo

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...