1. Ipossia indotta da CoCl 2 soluzione Cobalto (II) cloruro esaidrato (CoCl 2 • 6H 2 O, PM = 237,9) è un induttore chimico di ipossia-inducibile fattore-1.3 8. Questo prodotto è solubile in acqua (100 mg / ml), ottenendo una soluzione limpida e rosso. <ol><li> Preparare una soluzione 25mM stock in acqua sterile dd (preparare immediatamente prima dell&#39;uso) </li><li> Usa CoCl 2 alla concentrazione finale di 100μM nei vostri mezzi normali di coltura cellulare per indurre ipossia. </li><li> Aggiungere il CoCl 2 supporti contenenti alle vostre cellule e incubare le culture per 24 ore in un convenzionale incubatore (37 ° C, 5% 2 C0). </li></ol> La concentrazione di sopra lavori per le linee cellulari che abbiamo testato, ma ogni linea cellulare deve essere testato a diverse concentrazioni (per stabilire una curva dose-dipendente), così come in tempi di incubazione diversi al fine di limitare la tossicità legata alla droga ed ottimizzare il dosaggio. 2. L&#39;ipossia indotta a modulare Incubator Camera <ol><li> Preparare almeno due colture di cellule identiche (cultura gemello). Le colture cellulari possono essere in fusti, piatti o piatti della cultura. Per aprire la camera, per prima cosa aprire i due fascette di plastica bianca situato sul tubo attaccato alla camera (tubi utilizzati per l&#39;iniezione / eliminazione dei gas ipossia) e delicatamente aprire il morsetto ad anello in acciaio. </li><li> Rilasciare il morsetto. Il coperchio e vassoi possono essere rimossi. </li><li> Posizionare la coltura cellulare nella camera di ipossia. Anche mettere una piastra di Petri contenente acqua sterile nella camera di fornire un&#39;adeguata umidificazione delle culture. </li><li> Luogo della cultura &quot;gemello&quot; cella in normossia come controllo. </li><li> Assicurati che il tuo vassoi sono garantiti e non in movimento, e quindi chiudere la camera con il suo coperchio. Posizionare correttamente il morsetto anello in acciaio per garantire la chiusura ermetica della camera e chiuderlo. </li><li> Per creare l&#39;ipossia, collegare il tubo di un &quot;serbatoio di ipossia&quot; contenente un 1% O 2 miscela di gas. Se avete un misuratore di portata collegato al vostro serbatoio da camera saranno direttamente collegati ad esso (gas serbatoio-flow metri da camera). Usiamo un misuratore di portata incorporato nel nostro regolatore. </li><li> E &#39;importante rimuovere la maggior parte se non tutte ossigeno presente nella camera e nei vostri mezzi, per farlo lavare la camera aprendo il serbatoio del gas ad una portata di 20 litri al minuti per 7-10 minuti, poi spegnere rapidamente il il flusso del gas e chiudere completamente la camera chiudendo entrambe le fascette bianche. </li><li> Ritorna la camera ad una convenzionale incubatore per il periodo di tempo desiderato. Se si utilizzano grandi culture, consentire ai media nelle culture di de-gas per 1 - 2 ore e poi ripetere a filo. I passi sopra si basano sulle raccomandazioni costruttore &#39;(Billups-Rothenberg, Inc). Assicurati di serbatoio del gas siano garantiti a tutti i tempi. </li></ol> Note: <ul><li> Al fine di eliminare l&#39;O 2 contenute nei media è consigliato a ri-gas nella camera di una volta dopo 1-3 ore. </li><li> Per il tempo di incubazione più lungo di 48 ore è anche consigliato di re-gas della camera. </li><li> Sensori di ossigeno (elettrodi / manometro) che consentono misurazioni della O 2 contenuto in cellule / camera fornirà una misurazione più precisa del intracellulare / camera di O 2 contiene). </li><li> Cellule in coltura di varie lunghezze di tempo per fare una curva a tempo aiuterebbe determinare l&#39;espressione ottimale tempo di HIF-1α nelle vostre cellule particolare. </li></ul> 3. Valutazione di ipossia <ol><li> HIF-1 α rilevamento mediante western blot. <ol><li> Riaprire la tua camera come descritto nella sezione 2.2 e immediatamente inserire il vostro culture sul ghiaccio. </li><li> Lyse ipossia cellule trattate e non trattate con soluzione al 5% SDS nelle piastre quindi trasferire il lyzate cellula ai tubi, ultrasuoni, spindown i detriti cellulari e raccogliere il surnatante. </li><li> Misurare la concentrazione di proteine ​​con BCA kit, preparare campione con l&#39;aggiunta di tampone di caricamento e il riscaldamento a 95 ° C per 5 min. </li><li> Elettroforesi delle proteine ​​su un 8% SDS-poliacrilammide gel e il trasferimento alle membrane di nitrocellulosa. </li><li> Bloccare la membrana in PBS contenente 5% senza grassi del latte secco e lo 0,1% di Tween 20 a temperatura ambiente per 1 ora o a 4 ° C durante la notte su agitatore. </li><li> Durante la notte con anti-HIF-1α anticorpo primario monoclonale (1 / 600) nel buffer stesso immunoblot a 4 ° C. </li><li> Lavare 3 volte in PBS contenente 0,1% di Tween 20 a temperatura ambiente, 5 minuti ogni volta e incubare con anticorpi secondari HRP-(1 / 2000) in PBS + 0,1% Tween 20 per 45 min. </li><li> Lavare 3 volte in PBS contenente 0,1% di Tween 20 a temperatura ambiente, 5 min / ora. </li><li> Usa Pierce ECL kit e X-ray film per mostrare la band proteina </li></ol></li><li> L&#39;ipossia Responsive Element (HRE). HIF-1α regola numerosi geni (cioè VEGF, EPO) legandosi a una sequenza denominata ipossia elemento regolatore (HRE). Utilizzando HRE associati a un gene marcatore it è possibile rilevare l&#39;attività HIF 9. Per utilizzare questo metodo, in primo luogo è necessario trasfezione le cellule con un plasmide HRE-luciferasi utilizzando un metodo di trasfezione adattato alle vostre cellule. Per esempio abbiamo usato Lipofectamine 2000. Celle devono essere transfettate almeno 4 ore prima di essere incubate in ipossia e normossia. Questa volta permette al DNA di penetrare nelle cellule in modo che questo primo passo non è influenzata dall&#39;ipossia. Il segnale luciferasi viene rilevato utilizzando un kit di luciferasi Assay. <ol><li> Incubare il tuo HRE transfettate cellule in ipossia in camera di modulare o utilizzare HRE transfettate cellule incubate con CoCl 2, includere un controllo normossia. </li><li> Dopo il periodo di incubazione desiderato, togliere terreno di crescita dalle cellule. </li><li> Lavare le cellule in PBS, rimuovere la PBS. </li><li> Dispensare 400μl reagente 1X lisi nel piatto cultura e raschiare le cellule attaccato, poi trasferirli in una provetta da micro. </li><li> Detriti pellet per centrifugazione breve. </li><li> 20μl mix di lisati cellulari surnatante con 100μl di Reagente saggio luciferasi e misurare la luminescenza con un luminometro. </li></ol></li></ol> 4. Rappresentante dei risultati: In cellule K562 (linea cellulare di leucemia umana) in coltura 2 giorni in ossigeno, rispetto al normossia, ipossia induce un aumento di HIF-1α proteina che è stato rilevato dal western blot (Figura 1). Utilizzando HRE-luciferasi modificato 293 cellule (embrionali linea cellulare di rene) coltivate in ipossia, un significativo aumento di HIF-1α attività è stata rilevata nelle cellule ipossiche (Figura 2). <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Figura 1"/> Figura 1: Aumento HIF-1α nelle cellule ipossiche. K562 cellule sono state cultura in normossia e ipossia (camera di ipossia) per 48 ore e analizzati mediante western blot utilizzando un anticorpo specifico per HIF-1α. Un anticorpo specifico per l&#39;actina è stata utilizzata per il controllo di carico. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Figura 2"/> Figura 2: HIF-1α attività. HRE-luciferasi modificato 293 cellule sono state coltivate in ipossia e normossia per 48 ore e poi lisati di rilevare il segnale luciferasi utilizzando un kit luciferasi saggio e un luminometro. I risultati sono espressi come unità di luminescenza relativa (RLU).

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Proliferazione cellulare e vitalità in condizioni di ipossia variano molto a seconda dei tipi di cellule. Pertanto, si dovrebbe regolare il numero di cellulare o il numero di piatti culture si parte con per assicurarsi che si avrà abbastanza cellule / proteine ​​per i vostri esperimenti. Il metodo di cloruro di cobalto ha il vantaggio di essere poco costoso e veloce. Questo prodotto imita ipossia inducendo HIF-1/3α ma può anche regolare altri geni, assicurarsi che questo prodotto sia adatto al vostro progetto, controllando quali altri effetti potrebbe avere sulla funzione e il fenotipo delle cellule particolari indipendentemente dal &quot;mimare- ipossia &quot;effetto. Un altro farmaco usato anche per simulare l&#39;ipossia è deferoxamina mesilato (deferoxamina, impiegato a 100 concentrazione mM finale). L&#39;uso di questi farmaci consente al sperimentatore di aprire la piastra di coltura / piatto / fiasco molte volte senza compromettere la &quot;condizione di ipossia&quot;. Il livello di ossigeno contenuto nella miscela di gas può variare a seconda della esperimenti e tipi di cellule come valori ipossia variano a seconda dei tessuti e tipi cellulari. Infatti, alcune cellule ipossiche sono nel 5% O 2 bisogno di altri meno dell&#39;1% O 2 da ipossia. 5% di CO 2 è aggiunto alla miscela di gas per stabilizzare il pH delle culture, il resto del gas è di solito azoto. Camere ipossiche hanno il vantaggio di non utilizzare farmaci che possono alternare il comportamento delle cellule indipendentemente dalla tensione di ossigeno. Tuttavia, non tutti i tipi di sperimentazione può essere fatto come l&#39;ossigeno rientra nella camera ad ogni apertura e diminuisce così l&#39;ipossia. Si dovrebbe prendere in considerazione questo fattore nel vostro esperimenti; ipossia / riossigenazione è una specifica condizione che può colpire alcuni tipi di cellule. Un&#39;alternativa è quella di utilizzare postazioni ipossia (collegato al premiscelati serbatoi di gas o per sistemi di miscelazione dei gas) o più grande incubatore ipossia (camera di processo ipossia con vano portaoggetti) che permette la sperimentazione di cambiare i media e manipolare le cellule in continuo ambiente ipossico. Varie camere di ipossia sono stati commercializzati negli ultimi dieci anni e deve essere scelto in base al tuo laboratorio utilizzati, progetti, spazio, dimensione e budget. Sempre garantire il buon uso e la condizione della vostra camera per garantire condizioni di coltura corretto. In generale quando si utilizza una camera, il livello di ipossia può essere modulata scegliendo il mix di gas vari (es. 1%, 5% al ​​10% di ossigeno). Per quanto riguarda la rilevazione di HIF-1α, è importante sapere che alcune linee cellulari tumorali esprimere HIF-1α in normossia. E &#39;quindi fondamentale utilizzare un normossia-controllo per determinare il livello basale di HIF in queste linee cellulari. HIF-1α può anche essere presente in normossia non-maligne delle cellule in seguito alla stimolazione delle cellule o stress. Questo potrebbe verificarsi anche se queste cellule sono affamate, assicurarsi che il proprio colture di cellule di alimentazione siano correttamente e mantenuto.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Cobalto (II) cloruro </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> Incubatore Camera </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> HRE-luciferasi plasmide </td><td> Adgene </td><td> Plasmide 26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

L&#39;ipossia viene definita come la riduzione o la mancanza di ossigeno a organi, tessuti o cellule. Questa diminuzione di tensione di ossigeno può essere dovuta ad un apporto ridotto di ossigeno (cause includono insufficiente rete di vasi sanguigni, difettoso dei vasi sanguigni, e anemia) o ad un aumento del consumo di ossigeno rispetto alla fornitura (causata da un tasso di improvvisa proliferazione di cellule superiore) . L&#39;ipossia può essere fisiologico o patologico, come nei tumori solidi 1-3, artrite reumatoide, aterosclerosi, ecc ... Ogni tessuti e cellule hanno una diversa capacità di adattarsi a questa nuova condizione. Durante l&#39;ipossia, l&#39;ipossia inducible factor alpha (HIF) è stabilizzata e regolamenta vari geni come quelli coinvolti nell&#39;angiogenesi o il trasporto di ossigeno 4. La stabilizzazione di questa proteina è una caratteristica di ipossia, quindi il rilevamento HIF è utilizzato di routine per lo screening per 5-7 ipossia. In questo articolo proponiamo due semplici metodi per indurre ipossia in colture cellulari di mammifero e semplici test per valutare lo stato di queste cellule ipossiche.

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

Qui vi proponiamo semplici metodi per indurre ipossia in colture cellulari e semplici test per valutare lo stato di ipossia delle culture.

Induzione e test di ipossia in coltura cellulare

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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Research

JoVE Journal

Biology

84.4K Views.  Baylor College of Medicine. Qui vi proponiamo semplici metodi per indurre ipossia in colture cellulari e semplici test per valutare lo stato di ipossia delle culture.

Video: Induction and Testing of Hypoxia in Cell Culture

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura di cellule precursori staminali neurali del mouse

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Ricerca

Biologia

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Induzione e Monitoraggio adottivi di ipersensibilità ritardata nei ratti

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Il Metodo Culture Slice per seguire lo sviluppo dei germi dente Espianto Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, Cultura e trapianto di cellule muscolari satellite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

La proteina C-FOS immunoistologiche Detection: uno strumento utile come indicatore di percorsi centrali coinvolti nelle risposte fisiologiche specifiche<em&gt; In Vivo</em&gt; e<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Rilevamento di RNA-binding proteins by<em&gt; In Vitro</em&gt; RNA a tendina nella cultura degli adipociti

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Induzione e rilevamento dell'apoptosi in una cultura primaria delle cellule intestinali del cetriolo marino

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...