Vorremmo ringraziare CIRM per il supporto di questo lavoro. La linea cellulare è stata generosamente donata dal Dott. Andras Nagy al Mount Sinai Hospital, Toronto, Ontario.

1. Fare Shrinky-Mold Dink <ol><li> Stampare il modello voluto sulla Shrinky-dink foglio utilizzando una stampante buona definizione. </li><li> Cuocere Shrinky-dink foglio a 163 ° C per circa 10 minuti, o fino a completo ristretto e aver acquisito una forma regolare. </li><li> Dopo Shrinky-dink stampo è raffreddato, immergere in un bagno di isopropanolo fino a quando l&#39;intera superficie è a malapena coperto. </li><li> Attenzione, spray alcuni acetone sopra lo stampo e agitare il contenitore un paio di volte. Aggiungere più isopropanolo per lavare l&#39;eccesso di acetone e ripetere l&#39;operazione un paio di volte fino a quando Shrinky-muffa sembra pulito. </li><li> Stampo immergere in acqua distillata per 10 minuti per lavare la parte rimanente solvente organico. </li><li> Aria pulita Shrinky-stampo. Ri-calore per circa 5 minuti a 163 ° C. Questo inchiostro compatta e far evaporare i solventi rimanenti. </li></ol> 2. Fare PDMS pozzetti <ol><li> Preparare una miscela 10:01 agente PDMS / indurimento ed agitare vigorosamente per pochi minuti. </li><li> Luogo Shrinky-dink stampo in una piccola scatola di Petri. Versare il composto PDMS fino a raggiungere circa 1 / 2 cm su tutta la superficie dello stampo. </li><li> Piatto posto sotto campana sottovuoto per eliminare tutte le bolle dalla miscela PDMS. </li><li> Piatto posto in forno a 70 ° C, durante la notte. </li><li> Tagliare solido PDMS da muffe e associarlo a un vetrino semplicemente applicando una pressione. </li><li> Eliminare prima micropozzetti-chip, dal momento che ha i residui di inchiostro incrostato tra PDMS. </li><li> Ripetete questa procedura per produrre un secondo chip che è inchiostro libero e ha una forma più definita. </li><li> Pulire micropozzetti chip usando la soluzione di etanolo al 70%. Posto sotto fonte di luce UV per 10 minuti per sterilizzarlo. </li></ol> 3. Cellule cattura in pozzetti <ol><li> Contare le celle e diluire in terreni di coltura alla concentrazione desiderata (a seconda del numero delle cellule iniziali in pozzi che si desidera). Per esempio, per ottenere circa 10-15 cellule per pozzetto (media = 11, SD = 5.4, tasso di carico = 93%), abbiamo usato una concentrazione di 8 × 10 4 cellule / ml. Per una concentrazione di 17 × 10 4 cellule / ml, si potrebbe ottenere affidabile tra 25 e 35 cellule per pozzetto (media = 27,17857 DS = 7.7, tasso di carico = 100%). </li><li> Attentamente luogo di chip micropozzetti in un tubo da centrifuga da 50 ml contenente una base solidificato PDMS. </li><li> Aggiungere circa ml 2JDP della soluzione di cellula. </li><li> Centrifugare per 5 minuti a 760 giri al minuto e 4 ° C. </li><li> Pipettare in eccesso di soluzione e lavate accuratamente micropozzetti con la soluzione PBS 1X. </li><li> Mettere in un piatto micropozzetti Petry piccolo, facendo attenzione tenendo il chip dalla provetta. </li><li> Lavare l&#39;eccesso di cellule con 1 X soluzione PBS. </li><li> Luogo di chip micropozzetti sotto un microscopio invertito per verificare il numero previsto di cellule per pozzetto. </li><li> Incubare micropozzetti contenenti cellule in condizioni standard. <img src="/files/ftp_upload/old/692_2008_7_21_1/692_Figure-01.png" alt="Figura-01" width="350" height="183" /></li></ol> 4. Incubazione delle cellule <ol><li> Seguire il protocollo normale EB in laboratorio. </li><li> Cambia il medium lentamente dal lato della camera, non disturbare la cella nel micropozzetti. </li></ol>

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li><li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines:+Formation+of+visceral+yolk+sac,+blood+islands+and+myocardium.">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li><li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li><li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies.">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li><li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+differentiation+of+stem+cells.">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li><li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP/BMPR/Smad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media.">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li><li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li></ol>

Abbiamo sviluppato un metodo cultura semplice, rapida e scalabile per caricare predefiniti numero di cellule nei pozzetti microfabbricazione (stampato da Shrinky-Dinks) e mantenerli per lo sviluppo del corpo embrionali. Infine, queste cellule sono facilmente accessibili per ulteriori analisi e sperimentazione. Questo metodo è suscettibile di ogni laboratorio e non richiede attrezzature dedicate perché abbiamo ovviare alla necessità di fotolitografia. Siamo in grado di variare la dimensione dei pozzetti così come la concentrazione di cel...

Per i media Mesc (McCloskey laboratorio di protocollo): (per i media totale preparato: 50ml, 100ml) KO DMEM: 40.8ml; 81.6ml KSR 15%: 7.5ml; 15ml 1x Penn-Strep: 0,5 ml; 1ml 2mM L-glutammina: 0,5 ml; 1ml NEAA: 0,5 ml; 1ml LIF: 100ul; 200ul BMP-4 (10ng/ml): 50ul; 100ul diluito BME:. 50ul; 100ul (Aggiungi 35ul di sterile BME filtrato a 5 ml di PBS e siringhe filtro sterilizzare Scartare dopo 2 settimane. Concentrazione finale della soluzione è 0,1 mM)

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

Corpi embrionali (EB) sono aggregati di cellule staminali embrionali. Il modo più comune di creare questi aggregati è il metodo goccia, un approccio laborioso di pipettaggio un numero arbitrario di celle in piastre. Le interazioni tra le cellule staminali costretti a stretta vicinanza l&#39;uno dall&#39;altro favorisce la generazione di EBS. Perché i media in ciascuno dei pozzi deve essere manualmente scambiati ogni giorno, questo approccio è manualmente intensiva. Inoltre, poiché i parametri ambientali, tra cui cellula-cellula, cellula-interazioni tra fattori solubili, pH, ossigeno e la disponibilità possono essere le funzioni di taglia EB, popolazioni di cellule ottenute dalle tradizionali gocce può variare notevolmente, anche se colto in condizioni identiche. Recenti studi hanno infatti dimostrato che il numero iniziale di cellule che formano l&#39;aggregato possono avere effetti significativi sulla differenziazione delle cellule staminali. Abbiamo sviluppato un metodo cultura semplice, rapida e scalabile per caricare predefiniti numero di cellule nei pozzetti microfabbricazione e mantenerli per lo sviluppo del corpo embrionali. Infine, queste cellule sono facilmente accessibili per ulteriori analisi e sperimentazione. Questo metodo è suscettibile di ogni laboratorio e non richiede apparecchiature dedicate. Dimostriamo questo metodo mediante la creazione di corpi embrionali utilizzando una linea cellulare rosso fluorescente mouse (129S6B6-F1).

Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Mostriamo un metodo semplice e rapido per caricare predefiniti numero di cellule nei pozzetti microfabbricazione e mantenerli per lo sviluppo del corpo embrionali.

Shrinky-Dink Hanging Gocce: un modo semplice per forma e cultura corpi embrionali

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

shrinky-dink-hanging-drops-simple-way-to-form-culture-embryoid

Research

JoVE Journal

Biology

14.9K Views.  University of California Merced - UC Merced. Mostriamo un metodo semplice e rapido per caricare predefiniti numero di cellule nei pozzetti microfabbricazione e mantenerli per lo sviluppo del corpo embrionali.

Video: Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Ricerca

Biologia

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Analisi delle cellule staminali pluripotenti utilizzando criosezioni di corpi embrionali

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Tecnica semplice ed efficace per la preparazione delle sospensioni cellulari testicolari

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Metodo semplice per DNA fluorescenza<em&gt; In Situ</em&gt; Ibridazione di cromosomi Squashed

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Utilizzando il mouse cellule tumorali mammarie per insegnare concetti di biologia di base: un modulo di laboratorio semplice

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Un'analisi del Reporter di fluorescenza-basata semplice per identificare i componenti cellulari necessari per tossina ricina una catena (RTA) tratta di lievito

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Un metodo semplice per l'isolamento di protoplasti di soia e l'applicazione di analisi di espressione genica transitoria

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.