Finanziato dalla Sigma-Aldrich

1. Rilevamento micoplasma <ol><li> Surnatanti di coltura cellulare può essere verificata direttamente oppure il campione può predisposti per essere utilizzati in un secondo momento. Per preparare per un uso successivo posto 100 l di surnatante in un tubo di amplificazione sterile e incubare ° C per 5 minuti. A 95 Una volta che questo è completo il campione può essere conservato a 2-8 ° C per un massimo di una settimana. Poco prima di eseguire la centrifuga brevemente campione (5 secondi) per far sedimentare i detriti cellulari. </li><li> Per preparare i campioni per PCR, determinare il volume totale di Jumpstart Taq DNA polimerasi / reidratazione buffer necessaria per le reazioni. Ci prepareremo 5 reazioni totale. Cinque reazioni richiederà 2.5μl di Taq e 114.5μl di tampone di reidratazione. Questo conterrà un minimo di una unità di Taq per reazione e 22.5μl di tampone di reidratazione per reazione del campione e di controllo negativo, più 24.5μl di tampone di reidratazione per il controllo positivo. 1 unità di DNA polimerasi per reazione deve essere aggiunto al volume adeguato di tampone di reidratazione. Questo varia a seconda del Taq utilizzato. </li><li> Posizionare il volume calcolato di Taq DNA polimerasi in una provetta pulita e seguire con il volume calcolato di buffer di reidratazione. La polimerasi / reidratazione buffer di DNA deve essere mescolato delicatamente muovendo il tubo. Questa miscela non dovrebbero essere posti in agitazione. </li><li> Per preparare il controllo negativo e campioni di utilizzare i tubi di reazione trasparente fornito nel kit. I tubi di reazione forniti nel kit contengono già la nuclueotides, primer e DNA di controllo interno. 23 ml di mix Jumpstart di buffer Taq DNA polimerasi / reidratazione, così come redatto nei passi precedenti, deve essere posto in ciascuno dei controllo negativo e provette. Aggiungere 2 ml di acqua DNA libero per il controllo negativo e aggiungere 2 ml di campione per ognuna delle provette dei campioni e l&#39;etichetta. Mescolare il contenuto di sfogliando i tubi. Contenuti non devono essere posti in agitazione. </li><li> Per preparare il controllo positivo utilizzare i tubi di reazione rosa fornito nel kit. I tubi di reazione forniti nel kit contengono già la nuclueotides, primer e DNA di controllo interno. Aggiungere 25 ml di mix Jumpstart di buffer Taq DNA polimerasi / reidratazione, così come redatto nei passaggi precedenti, per i tubi di reazione e l&#39;etichetta. Mescolare il contenuto di sfogliando i tubi. Contenuti non devono essere posti in agitazione. Incubare il controllo negativo, controllo positivo e provette a temperatura ambiente per 5 minuti. </li><li> Per la PCR che si terrà i campioni devono essere collocati in un termociclatore. Quando si utilizza JumpStart Taq un passo di attivazione non è necessario. Posizionare le provette nel termociclatore. I cicli deve essere impostato come segue: 94 ° C per 30 secondi, 55 ° C per 30 secondi e 72 ° C per 40 secondi. Questi cicli verrà eseguito 40 volte. </li><li> Una volta che i cicli di PCR hanno completato i campioni devono essere raffreddata a 4-8 ° C per rimuoverli dal termociclatore e mettendo in ghiaccio. Quando i campioni si sono raffreddati essi dovrebbero essere caricati sul gel di agarosio per l&#39;elettroforesi. Gel di carico e coloranti non sono necessari in quanto sono già presenti nei tubi di reazione. Direttamente carico di 8 microlitri per ogni PCR in una corsia separata. Fermare il elettroforesi dopo la migrazione di 2,5 - 3,0 centimetri. </li></ol> 2. Rappresentante Risultati Il controllo negativo dovrebbe mostrare una banda a circa 481 bp. Questo può essere visto nella colonna di controllo negativo sul gel elettroforesi che viene mostrato all&#39;inizio del video. L&#39;assenza della banda di controllo negativo a 481 bp è un buon indicatore che l&#39;attività della polimerasi non era sufficienttaq utilizzato non era abbastanza sensibile. Mycoplasma campioni positivi mostrano bande nel range di 270 ± 8 bp. La band sarà pesante per i campioni altamente contaminati e debole per i campioni leggermente contaminati. Le variazioni di intensità può essere visto nelle colonne campione positivo sul gel elettroforesi che viene mostrato all&#39;inizio del video. Tutti i campioni contengono un controllo interno negativo per dimostrare che la PCR si è verificato come previsto. E &#39;normale vedere l&#39;assenza del controllo interno su campioni altamente contaminati. Se non c&#39;è una banda nel range positivo o una band in campo negativo sul vostro campione questo è un buon indicatore che i campioni sono inibite. Se il campione è inibito il inibitori della PCR può essere rimosso effettuando una estrazione del DNA. <img alt="Figura 1" src="/files/ftp_upload/3057/3057fig1.jpg" /> Bande Amplicon Figura 1 rilevanti:. Risultati di rilevare micoplasma mediante PCR sono mostrati. Corsia 2 è il controllo negativo, che dovrebbe mostrare una banda di circa 481 bp. L&#39;assenza della banda di controllo negativo a 481 bp è un buon indicatore che la Taq utilizzato non era abbastanza sensibile. Mycoplasma campioni positivi mostrerà bande nel range di 270 + 8 bp come mostrato in corsie 3 - 6. La band sarà heavy per campioni altamente contaminati (corsia 6) e leggermente debole per i campioni contaminati (corsia 4). Tutti i campioni contengono un controllo interno negativo (481 bp) per dimostrare che la PCR si è verificato come previsto. E &#39;normale vedere l&#39;assenza del controllo interno su campioni altamente contaminati.

<ol>
	<li>Wirth, M., Hauser, H., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+Sensitivity+of+Polymerase+Chain+Reaction+(PCR)+in+Comparison+with+Other+Methods+for+the+Detection+of+Mycoplasma+Contamination+in+Cell+Lines.">Specificity and Sensitivity of Polymerase Chain Reaction (PCR) in Comparison with Other Methods for the Detection of Mycoplasma Contamination in Cell Lines.</a> Journal of Immunological Methods. 164, 91-100 (1993).</li><li>Kong, H., Volokhov, D. V., George, J., Ikonomi, P., Chandler, D., Anderson, C., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+Cell+Culture+Enrichment+for+Improving+the+Sensitivity+of+Mycoplasma+Detection+Methods+Based+on+Nucleic+Acid+Amplification+Technology+(NAT).">Amplification of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT).</a> Applied Microbiology and Biotechnology. 77, 223-232 (2007).</li><li>Volokhov, D. V., Graham, L. J., Borson, K. A., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+Testing+of+Cell+Substrates+and+Biologics:+Review+of+Alternative+Non-Microbiological+Techniques.">Mycoplasma Testing of Cell Substrates and Biologics: Review of Alternative Non-Microbiological Techniques.</a> Molecular and Cellular Probes. , (2011).</li></ol>

Gli autori sono dipendenti di Sigma-Aldrich che ha prodotto i reagenti e strumenti utilizzati in questo articolo.

A causa della estrema sensibilità del kit, se eseguita correttamente il kit devono fornire risultati precisi che indicano se il campione è la contaminazione da micoplasma. Il kit è stato ottimizzato per l&#39;utilizzo con JumpStart Taq DNA polimerasi. Taqs altri possono essere utilizzati nella preparazione dei campioni, ma la banda di controllo interno a 481 bp dovrebbe essere presente per indicare la sensibilità corretta l&#39;alternativa Taq. E &#39;possibile per le bande positivo ad esporre densità diverse a seconda del livello di contaminazione. E &#39;possibile osservare l&#39;assenza della banda di controllo interno su campioni fortemente contaminati. Usando cautela e tecnica adeguata per la preparazione dei campioni per evitare la contaminazione sono cruciali per il corretto. quando si utilizza il rilevamento LookOut Mycoplasma Detection Kit PCR.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> LookOut Mycoplasma PCR Detection Kit </td><td> Sigma-Aldrich </td><td> MP0035 </td><td> </td></tr><tr><td> JumpStart Taq DNA polimerasi </td><td> Sigma-Aldrich </td><td> D9307 </td><td> </td></tr><tr><td> GenElute Sangue kit del DNA genomico </td><td> Sigma-Aldrich </td><td> MP0030 </td><td> </td></tr><tr><td> Tubi di amplificazione Nunc </td><td> Sigma-Aldrich </td><td> T0447 </td><td> </td></tr></tbody></table>

optimized pcr-based detection of mycoplasma

Il mantenimento di contaminazione senza linee di cellule è indispensabile per cellulari basati su ricerche. Tra le maggiori preoccupazioni dei contaminanti sono la contaminazione da micoplasma. Anche se micoplasma di solito non uccidono le cellule contaminate, sono difficili da rilevare e possono causare una serie di effetti sulle cellule in coltura, tra cui alterato metabolismo, rallentato proliferazione e aberrazioni cromosomiche. In breve, la contaminazione da micoplasma compromette il valore di tali linee cellulari nel fornire dati accurati per la ricerca delle scienze della vita. Le fonti di contaminazione da micoplasma in laboratorio sono molto difficili da controllare completamente. Come alcune specie di micoplasmi sono presenti sulla cute umana, possono essere introdotte attraverso povero tecnica asettica. Inoltre, possono venire dagli integratori contaminati come siero fetale bovino e, soprattutto, dalle altre colture di cellule contaminate. Una volta micoplasma contamina una cultura, può rapidamente diffondersi di contaminare altre aree del laboratorio. Rispetto rigoroso di buone pratiche di laboratorio, come buona tecnica asettica sono fondamentali, e di routine per il micoplasma è altamente consigliato per un controllo adeguato della contaminazione da micoplasma. Basati sulla PCR rilevazione di micoplasmi è diventato un metodo molto popolare per la manutenzione ordinaria linea cellulare. Basati sulla PCR metodi di rilevamento sono altamente sensibili e in grado di fornire risultati rapidi, che permette ai ricercatori di rispondere rapidamente ad isolare ed eliminare la contaminazione una volta che viene rilevato in confronto al tempo necessario utilizzando tecniche microbiologiche. Il LookOut Mycoplasma Detection Kit PCR è molto sensibile, con un limite di rilevazione di soli 2 genomi per microlitri. Approfittando della DNA polimerasi altamente specifici JumpStart Taq e un design fondo di proprietà, i falsi positivi sono notevolmente ridotte. Il comodo formato 8-tubo, strisce pre-rivestiti con dNTPs e primers associati aiuta ad aumentare la produttività per soddisfare le esigenze dei clienti con i più grandi collezioni di linee cellulari. Data l&#39;estrema sensibilità del kit, grande cura deve essere presa per evitare la contaminazione accidentale di campioni e reagenti. Il passo-passo protocollo dimostriamo mette in evidenza le precauzioni e le pratiche necessarie per il rilevamento affidabile micoplasma. Abbiamo anche mostrare e discutere i risultati tipici e la loro interpretazione. Il nostro obiettivo è quello di garantire il successo di ricercatori che utilizzano il LookOut Mycoplasma Detection Kit PCR.

Watch this Scientific Journal Video about Optimized PCR-based Detection of Mycoplasma at JoVE.com

Optimized PCR-based Detection of Mycoplasma

Il LookOut Mycoplasma Detection Kit PCR sfrutta la reazione a catena della polimerasi (PCR), che è stabilito come metodo di scelta per la massima sensibilità nella rilevazione di contaminazione Mycoplasma, Acholeplasma e Ureaplasma in colture cellulari e di colture cellulari di altri derivati ​​biologici.

Ottimizzati basati sulla PCR Rilevazione di Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

optimized-pcr-based-detection-of-mycoplasma

Research

JoVE Journal

Biology

53.5K Views.  Sigma-Aldrich. Il LookOut Mycoplasma Detection Kit PCR sfrutta la reazione a catena della polimerasi (PCR), che è stabilito come metodo di scelta per la massima sensibilità nella rilevazione di contaminazione Mycoplasma, Acholeplasma e Ureaplasma in colture cellulari e di colture cellulari di altri derivati ​​biologici.

Video: Optimized PCR-based Detection of Mycoplasma

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Ottimizzato fibrina Gel Bead Assay per lo studio della angiogenesi

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ricerca

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Rilevamento di proteine ​​Ubiquitinazione

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Rilevamento delle metalloproteinasi della matrice funzionale di zimografia

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Un protocollo ottimizzato per la cura dei Fopius arisanus, Un parassitoide di frutta Tephritid mosche

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Impianto degli innesti vascolari elettrofilate con struttura ottimizzata in un modello del ratto

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Ottimizzato Scratch test per test In Vitro della migrazione cellulare con una fotocamera ottica automatizzata

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Semplice rilevamento delle ciglia primarie per immunofluorescenza

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Rilevamento e analisi automatizzati dell'esocitosi

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...