Il nostro lavoro è supportato dal NIH concede NS048447 e AG017216to KD. Li Liu desidera ringraziare il Dott. Heikki Tanila (Università di Kuopio, Kuopio, Finlandia) per la sua supervisione e supporto durante lo sviluppo del protocollo originale.

Tirare il tubo capillare di vetro <ol><li> Il tubo capillare di vetro viene acquistato dallo strumento Sutter Inc (vetro borosilicato, B100-75-10). </li><li> Tirare i tubi capillari su un Sutter P-87 Flaming micropipetta estrattore, con l&#39;indice di calore fissato a 300 e l&#39;indice di pressione fissato a 330. </li><li> Tagliare la punta del tubo capillare di vetro con le forbici, in modo che la punta conica ha un diametro interno di circa 0,5 mm. </li></ol> Cisterna magna tecnica di puntura di CSF Campioni di CSF sono prese dal cisterna magna (Figura 1) utilizzando un metodo che è stato pubblicato in precedenza 1. <img src="/files/ftp_upload/old/960_2008_10_13_2/960_Figure-01.png" alt="Figura 1" width="432" height="184" /> Figura 1 1. I topi sono anesthesized da ketamina (100mg/kg) e xilazina (10mg/kg), somministrato per via intraperitoneale. Durante il periodo di induzione dell&#39;anestesia, i topi sono conservati in un incubatore o 37 ° C. 2. La pelle del collo è rasata, e il mouse viene posto prono sullo strumento stereotassico con il contatto diretto di una piastra elettrica. Una sonda di temperatura rettale viene inserito nel retto modo che il calore prodotto dal pad di riscaldamento viene regolata in risposta ai cambiamenti della temperatura corporea. La testa è assicurato con le schede di testa. Il sito chirurgico è tamponata con iodio povidone al 10%, seguita da 7 0% di etanolo (ripetere 3 volte), e un&#39;incisione sagittale della pelle è fatta inferiore all&#39;occipite. 3. Sotto il microscopio dissezione, il tessuto sottocutaneo e dei muscoli (m. biventer del collo e m. retto capitis dorsale principale) sono separati per via smussa con una pinza. Un paio di microretractors è utilizzato per tenere i muscoli a pezzi. 4. Il mouse è stabilito in modo che la testa forma un angolo di quasi 135 ° con il corpo. 5. Sotto il microscopio dissezione, la dura madre della cisterna magna appare come un triangolo scintillante e chiara inverso attraverso il quale il midollo allungato e di un vaso sanguigno principale (arteria spinale dorsale), e lo spazio CSF ​​sono visibili (Figura 2). <img src="/files/ftp_upload/old/960_2008_10_13_3/960_Figure-02.png" alt="Figura 2" width="304" height="307" /> Figura 2 6. La dura madre è asciugate con tampone di cotone sterile. Penetrare il tubo capillare in magna Citerna attraverso la dura madre, laterale alla arteria spinale dorsale (Figura 2). A seguito di un notevole cambiamento nella resistenza per l&#39;inserimento del tubo capillare, il QCS scorre nel tubo capillare. 7. Rimuovere con attenzione il tubo capillare, e collegarlo a una siringa da 3 ml con un tubo di polietilene che ha un diametro interno 1 mm. Iniettare il liquido cerebrospinale in un pre-segnato 0,5 ml di tubo Eppendorf, e congelare immediatamente la provetta in ghiaccio secco e poi trasferirlo in un congelatore C ° -80. 8. Dopo il campionamento CSF, i muscoli sono nuovamente allineati, e la pelle viene suturata (4-0, Ethicon, Johnson &amp; Johnson). Circa 1 ml di NaCl 0,9% viene iniettata per via sottocutanea per evitare de-idratazione. Il mouse è conservato presso l&#39;incubatore per mantenere la temperatura corporea fino a che non recupera, il peso del mouse viene monitorata 1 giorno, e 1 settimana dopo l&#39;intervento chirurgico. Risultati e discussione Abbiamo descritto un protocollo altamente affidabile per il campionamento di serie del CSF da topi, senza contaminazioni plasma rilevabili. 1. L&#39;intera procedura richiede solitamente 10 min per il mouse (incluso anestesia). Il volume di CSF ottenuti dipende dal ceppo di topi. Nella PS / APP topi doppio Tg abbiamo usato 2, il volume medio è di circa 5 microlitri (3-7 mL), mentre la (JNPL3) topi P301L 3 danno una resa di circa 10 microlitri-15 microlitri. Per il campionamento di serie, un massimo di 7-8 microlitri può essere tranquillamente assunto di volta in volta ad un intervallo di 2-3 mesi. 2. Durante l&#39;intervento, è importante posizionare la testa e il corpo del mouse correttamente sul telaio stereotassico in modo che la dura madre della cisterna magna può essere esposto a sufficienza. Accuratamente evitare i vasi sanguigni quando penetra la dura madre con il tubo capillare per evitare la contaminazione da proteine ​​plasmatiche, che possono essere monitorate nel campione di liquido cerebrospinale di immunoblotting per ApoB 4. 3. Riducendo al minimo il danno ai tessuti durante ogni intervento chirurgico è importante, come conglutination tessuto può aumentare la difficoltà di sanguinamento predisponendo per il campionamento successivo. 4. La temperatura corporea deve essere ben mantenuto durante l&#39;intervento chirurgico a causa ipotermia causata da anestesia possono influenzare significativamente i livelli di tau fosforilata nel cervello molto rapidamente 5. 5. L&#39;Ab ELISA protocollo e gli anticorpi che abbiamo utilizzato sono stati descritti in dettaglio nel Refolo et al. 6. Due ml di liquor dal PS / APP topi darà risultati soddisfacenti se diluito 1:50-1:60. Per l&#39;ELISA tau, 4-5 CSF microlitri da P301L (JNPL3) topi sarà sufficiente per larilevazione del totale tau, o tau fosforo ylated a treonina 231 (I kit sono acquistati da Bio-Source). 6. Questa tecnica può essere applicata ad altri modelli murini di patologie neurologiche, per la quale un piccolo volume di CSF è soddisfacente per le analisi desiderata.

<ol>
	<li>Liu, 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=Longitudinal+observation+on+CSF+Abeta42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF Abeta42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol Dis. 17, 516 (2004).</li><li>Holcomb, 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=Accelerated+Alzheimer-type+phenotype+in+transgenic+mice+carrying+both+mutant+amyloid+precursor+protein+and+presenilin+1+transgenes.">Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.</a> Nat Med. 4, 97 (1998).</li><li>Lewis, J., 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=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25, 402 (2000).</li><li>DeMattos, R. 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=Plaque-associated+disruption+of+CSF+and+plasma+amyloid-beta+(Abeta)+equilibrium+in+a+mouse+model+of+Alzheimer's+disease.">Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.</a> J Neurochem. 81, 229 (2002).</li><li>Planel, E., 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=Anesthesia+leads+to+tau+hyperphosphorylation+through+inhibition+of+phosphatase+activity+by+hypothermia.">Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.</a> J Neurosci. 27, 3090 (2007).</li><li>Refolo, L. M., 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=A+cholesterol-lowering+drug+reduces+beta-amyloid+pathology+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 8, 890 (2001).</li></ol>

Abbiamo descritto un protocollo altamente affidabile per il campionamento di serie del CSF da topi, senza contaminazioni plasma rilevabili. 1. L&#39;intera procedura richiede solitamente 10 min per il mouse (incluso anestesia). Il volume di CSF ottenuti dipende dal ceppo di topi. Nella PS / APP topi doppio Tg abbiamo used2, il volume medio è di circa 5 microlitri (3-7 mL), mentre la (JNPL3) topi P301L 3 danno una resa di circa 10 microlitri-15 microlitri. Per il campionamento di serie, un massimo di 7-8 microlitri può essere tranq...

a technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse 

La malattia di Alzheimer (AD) è una malattia neurodegenerativa progressiva che è patologicamente caratterizzata dalla deposizione extracellulare del peptide β-amiloide (Aβ) e l&#39;accumulo di intraneuronale iperfosforilata proteina tau. A causa del liquido cerebrospinale (CSF) è in contatto diretto con lo spazio extracellulare del cervello, fornisce un riflesso dei cambiamenti biochimici nel cervello in risposta a processi patologici. CSF di pazienti dC mostra una diminuzione del 42 aminoacidi forma di Aβ (Aβ42), e aumenta in totale iperfosforilata tau e tau, anche se i meccanismi responsabili di questi cambiamenti non sono ancora pienamente compreso. Transgenici (Tg) modelli murini di AD forniscono un&#39;eccellente opportunità per indagare come e perché i livelli di Aβ o tau nel liquido cerebrospinale cambiare con la progressione della malattia. Qui, dimostrano una raffinata tecnica di puntura cisterna magna per il campionamento CSF ​​dal mouse. Questa tecnica di campionamento estremamente delicata permette seriale campioni di liquido cerebrospinale di ottenere dal mouse stesso a intervalli di 2-3 mesi, che riduce sensibilmente l&#39;effetto confondente della tra-mouse variabilità nei livelli di Aβ o tau, che consente di rilevare alterazioni sottili nel tempo. In combinazione con Aβ e tau ELISA, questa tecnica sarà utile per studi volti a indagare il rapporto tra i livelli di CSF Aβ42 e tau, e il loro metabolismo nel cervello in modelli murini dC. Studi nei topi Tg potrebbe fornire la convalida importante per il potenziale di Aβ CSF o livelli di tau per essere utilizzati come marcatori biologici per monitorare la progressione della malattia, e per monitorare l&#39;effetto degli interventi terapeutici. Come i topi possono essere sacrificati e il cervello possono essere esaminati per cambiamenti biochimici o istologici, i meccanismi alla base dei cambiamenti CSF può essere meglio valutata. Questi dati sono suscettibili di essere informativo per l&#39;interpretazione dei cambiamenti umani CSF dC.

Watch this Scientific Journal Video about A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse at JoVE.com

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenici (Tg) modelli murini di AD forniscono un&#39;eccellente opportunità per indagare come e perché i livelli di Aβ o tau nel liquido cerebrospinale cambiare il progredire della malattia nei pazienti umani. Qui, dimostrano una raffinata tecnica di puntura cisterna magna per il campionamento CSF ​​seriale dal mouse.

Una tecnica per la raccolta seriale del liquido cerebrospinale dalla Magna Cisterna in Mouse

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

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Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

100.9K Views.  Columbia University.  Transgenici (Tg) modelli murini di AD forniscono un'eccellente opportunità per indagare come e perché i livelli di Aβ o tau nel liquido cerebrospinale cambiare il progredire della malattia nei pazienti umani. Qui, dimostrano una raffinata tecnica di puntura cisterna magna per il campionamento CSF ​​seriale dal mouse. 

Video: A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Isolamento del Vampiro di liquido extracellulare da<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Ricerca

Biologia

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Arricchimento di serie staminali spermatogoni e cellule progenitrici (SSC) in Cultura per la derivazione di lungo termine Lines topo adulto SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento dell&#39;RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La tecnica infiltrazione-centrifugazione per l&#39;estrazione di apoplastico fluido da foglie delle piante Utilizzo<em&gt; Phaseolus vulgaris</em&gt; Come un esempio

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblea e l&#39;applicazione di &quot;Shear Anelli &#39;: A Novel endoteliale modello per Orbital, unidirezionale e periodica del fluido di flusso e lo stress di taglio

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La tecnica del rene perfusi mouse isolato

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Raccolta a cella singola di cellule di trofoblasto in peri-impianto Stadio Embrioni Umani

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Raccolta dei tessuti ed estrazione dell'RNA dall'articolazione del ginocchio osteoartritico umano

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...