Vorremmo ringraziare Jonathan Cooper Lab Research Fred Hutchinson Cancer Centro per il permesso di usare il loro criostato. Questo lavoro è stato sostenuto da NIH e il WM Keck Foundation. BM è un collega Gordon and Betty Moore Foundation di Life Science Research. Siamo grati a Daniel Gottschling per aver condiviso il suo protocollo di fissaggio con noi.

1. Fissaggio Comunità lievito <ol><li> Crescono comunità lievito su un filtro a membrana (ad esempio, Millipore MF membrana filtrante; Figura 2A). Questo protocollo corrisponde ad una tipica comunità inferiore a 2x10 8 celle di un millimetro di diametro 6 filtro a membrana. </li><li> Utilizzare un pezzo di carta da filtro Whatman 541 per coprire il fondo di un diametro di 1 cm-ben (Figura 2B). Questa carta Whatman sarà utilizzato come vettore per trasferire la comunità in fasi successive. Aggiungere 1 ml di metanolo 100% al pozzo, e lasciare il pozzo a -20 ° C per la temperatura di equilibrarsi. </li><li> N 2 liquido messo in un dewar. Congelare comunità in azoto liquido per immersione della comunità per almeno 15 secondi in un liquido N 2 utilizzando una pinzetta (Figura 2C). </li><li> Trasferire comunità congelato al pozzo di -20 ° C metanolo (Figura 2D). Assicurarsi che la comunità sia tenuta in orizzontale quando si immerge nel metanolo.Attendere 20 min. Il filtro a membrana MF inizia a disintegrarsi in metanolo. </li><li> Trasferire comunità utilizzando il vettore 541 carta da filtro Whatman dal metanolo bene ad un piatto freddo Petri tenuta a -20 ° C per evaporazione metanolo (Figura 2E). Durante il trasferimento, tenere la comunità sulla parte superiore del supporto filtro Whatman e pareggio ospiti gocce in più di metanolo con un altro pezzo di spessa carta Whatman prima di mettere nel piatto freddo Petri. Ciò farà sì che il tempo di evaporazione è coerente da campione a campione. </li><li> Attendere 4-6 ore per l&#39;evaporazione finché il supporto del filtro Whatman sembra asciutto. Evaporazione insufficiente lascia metanolo residuo che interferisce con sezionamento. Oltre ad asciugatura crepe cause e deformazioni nelle comunità. </li><li> Prelevare il campione di congelatore a -20 ° C. Tagliare Whatman 541 carta da filtro non coperti dalla comunità. </li><li> Fare un cubo rettangolare pellicola di alluminio contenitore che è abbastanza grande in modo tale che il campione ha almeno ~ 2 mm dal margineciascun lato (Figura 2F). Posizionare la comunità sulla superficie inferiore della pellicola di alluminio contenitore, coprire immediatamente il campione con Office per 3 ~ 4 mm al di sopra dell&#39;altezza della comunità. Se importante, l&#39;orientamento del campione può essere regolata in questa fase in relazione ai lati del contenitore. Attendere 10 min. </li><li> Posizionare il PTOM-embedded campione su una piastra metallica in ghiaccio secco per congelare. Conservare le comunità congelati a -20 ° C o -80 ° C congelatore. </li></ol> 2. Sezionamento Comunità congelati per fluorescenza Imaging <ol><li> Impostare la temperatura della camera di cryosection a -25 ° C e lasciare i campioni nella camera finché la sua temperatura raggiunge la temperatura della camera. </li><li> Montare il campione su un crio-mount dopo l&#39;applicazione di una goccia di ottobre sul monte. Diffondere ottobre uniformemente sul lato del campione esposto con carta da filtro Whatman (fondo del contenitore) in modo che lo spessore ottobre è 2-3 mm. Lasciate congelare ottobre all&#39;interno della camera di criostato prima sezionamento. Garantire un margine ottobre di 2-3 mm su tutti i lati del campione contribuisce a preservare l&#39;integrità delle sezioni. </li><li> I lati del blocco campioni ottobre può essere utilizzato per allineare il campione. Se il campione non è allineato rispetto alla lama, sezionamento inizia da un angolo o lato del campione. Regolare il montaggio in modo che la lama taglia perfettamente verticale (o orizzontale) faccia del blocco campione. </li><li> Sezioni tipiche per le comunità di lievito sono ~ 14 micron di spessore. Dopo il taglio di una sezione, utilizzare un vetrino conservato a temperatura ambiente per raccogliere sezione, lentamente avvicinando la slitta nella sezione di taglio. La sezione si scioglierà e trasferimento al vetrino da microscopio. Fino a 18 sezioni possono essere trasferiti su una diapositiva. </li><li> Conservare i vetrini a basse temperature, preferibilmente -20 ° C, prima di imaging. Perdita di segnale è minimo quando conservata per una settimana a 4 ° C. Per il massimo segnale di fluorescenza, l&#39;imaging è preferibilmente effettuata subito dopo il sezionamento. </li></ol>

<ol>
	<li>Tolker-Nielsen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Microbial+Biofilm+Communities.">Spatial Organization of Microbial Biofilm Communities.</a> Microbial Ecology. 40 (2), 75-84 (2000).</li><li>Moller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Gene+Expression+in+Mixed-Culture+Biofilms:+Evidence+of+Metabolic+Interactions+between+Community+Members."> In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members.</a> Appl. Environ. Microbiol. 64 (2), 721-732 (1998).</li><li>Lepp, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanogenic+Archaea+and+human+periodontal+disease.">Methanogenic Archaea and human periodontal disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6176-6181 (2004).</li><li>V&#225;chov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environmental Microbiology. 11 (7), 1866-1877 (2009).</li><li>Min&#225;rikov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Gene+Expression+in+Cells+within+Yeast+Colonies.">Differentiated Gene Expression in Cells within Yeast Colonies.</a> Experimental Cell Research. 271 (2), 296-304 (2001).</li><li>Shou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cooperation+in+engineered+yeast+populations.">Synthetic cooperation in engineered yeast populations.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1877-1882 (2007).</li><li>Momeni, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+generates+a+unique+spatial+signature+in+microbial+communities.">Cooperation generates a unique spatial signature in microbial communities.</a> , (2012).</li><li>Kiernan, J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological and Histochemical Methods: Theory and Practice. , 4 (2008).</li><li>Piccirillo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+Pathway+and+Alkaline+pH+Regulate+Pattern+Formation+in+Yeast+Colonies.">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies.</a> Genetics. 184 (3), 707-716 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

La procedura qui presentata offre un modo per controllare la struttura interna delle comunità lievito. La fase di fissaggio metanolo rende la colonia di lievito rigido 8 e preserva l&#39;integrità della colonia, tuttavia, causa anche restringimento 8 che deve essere preso in considerazione nell&#39;interpretazione dei risultati. Noi di solito vedere circa il 30% in altezza ritiro delle colonie di lievito, rispetto alle colonie direttamente incorporati nei PTOM senza fissare 9. <p c...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> 100% Metanolo </td><td> JT Baker </td><td> 9070-01 </td><td></td></tr><tr><td> Tissue-Tek ottobre Compound </td><td> E vinci la scientifica </td><td> 4583 </td><td></td></tr><tr><td> Whatman Grade Paper No. 541 filtro quantitativa </td><td> Whatman </td><td> 1541-047 </td><td></td></tr><tr><td> Millipore MF-filtro a membrana </td><td> Millipore </td><td> HAWP04700 </td><td></td></tr><tr><td> Criostato </td><td> Leica </td><td> CM 1510 S </td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

I microbi in genere vivono in comunità. L&#39;organizzazione spaziale delle cellule all&#39;interno di una comunità si crede di influenzare la sopravvivenza e la funzione della comunità 1. Tecniche ottiche di sezionamento, tra cui la microscopia confocale e due fotoni, si sono dimostrati utili per osservare l&#39;organizzazione spaziale delle comunità batteriche e archaeal 2,3. Una combinazione di imaging confocale e sezionamento fisico di colonie di lievito ha rivelato organizzazione interna delle cellule 4. Tuttavia, diretto sezionamento ottico usando la microscopia confocale a due fotoni o è stato solo in grado di raggiungere un paio di strati di cellule in profondità in colonie di lievito. Questa limitazione è probabilmente a causa della dispersione di luce intensa da cellule di lievito 4. Qui vi presentiamo un metodo basato sulla determinazione e criosezionamento per ottenere la distribuzione spaziale delle cellule fluorescenti all&#39;interno delle comunità di Saccharomyces cerevisiae. Usiamo metanolo come agente fissativoda mantenere la distribuzione spaziale delle cellule. Comunità fissi sono infiltrati con OCT, congelati, e cryosectioned in un criostato. Imaging di fluorescenza delle sezioni rivela come l&#39;organizzazione interna delle cellule fluorescenti all&#39;interno della comunità. Esempi di comunità costituite ceppi di lievito che esprimono proteine ​​fluorescenti rosse e verdi dimostrare le potenzialità del metodo criosezionamento per rivelare la distribuzione spaziale delle cellule fluorescenti così come quella di espressione genica all&#39;interno colonie di lievito 2,3. Anche se la nostra attenzione si è concentrata sulle comunità di Saccharomyces cerevisiae, lo stesso metodo può potenzialmente essere applicato a esaminare altre comunità microbiche.

Immagini di fluorescenza di sezioni verticali delle comunità lievito contenenti rosso o verde etichettato popolazioni competitivi 6 sono mostrati nella Figura 3. Queste comunità sono costituiti da due ceppi di lievito e prototrofici loro competizione per risorse condivise, incluso lo spazio, porta a colonnare modelli 7, come mostrato nelle sezioni verticali. Risoluzioni fino a una singola cellula può essere risolto in sezioni. Figura 3 confronta sezioni delle co...

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Vi presentiamo un protocollo per congelare e criosezionamento comunità di lievito per osservare i modelli interni di cellule fluorescenti. Il metodo si basa su di fissaggio e metanolo-OCT-incorporamento per preservare la distribuzione spaziale di celle senza inattivare proteine ​​fluorescenti all&#39;interno di una comunità.

Criosezionamento Comunità di lievito per l&#39;esame di pattern di fluorescenza

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

cryosectioning-yeast-communities-for-examining-fluorescence-patterns

Research

JoVE Journal

Biology

8.8K Views.  Fred Hutchinson Cancer Research Center. Vi presentiamo un protocollo per congelare e criosezionamento comunità di lievito per osservare i modelli interni di cellule fluorescenti. Il metodo si basa su di fissaggio e metanolo-OCT-incorporamento per preservare la distribuzione spaziale di celle senza inattivare proteine ​​fluorescenti all'interno di una comunità.

Video: Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Flash Congelamento e criosezionamento E12.5 cervello di topo

Ricerca

Biologia

Microbial Communities in Nature and Laboratory - Interview

Comunità microbica in natura e laboratorio - Intervista

Biology of Microbial Communities  - Interview

Biologia delle comunità microbiche - Intervista

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Un dispositivo a microfluidi con pattern Groove per studiare il comportamento cellulare

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un saggio microscopia a fluorescenza per Mitophagy monitoraggio nel lievito Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativa vivo cellulare-microscopio a fluorescenza Analisi di lievito Fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

L&#39;acquisizione di fluorescenza filmati time-lapse di lievito in erba e Analisi Dinamica singola cella utilizzando innesti

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Metodo criosezionamento per Microdissezione di Murine colon mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...