Name: induction and testing of hypoxia in cell culture
Uploaded: 2011-08-12T13:00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

1. Hypoxie veroorzaakt door CoCl 2-oplossing Kobalt (II) Chloride hexahydraat (CoCl 2 • 6H 2 O, MW = 237,9) is een chemische inductor van hypoxie-induceerbare factor-1.3 8. Dit product is oplosbaar in water (100 mg / ml), waardoor een heldere, rode oplossing. <ol><li> Bereid een 25mm voorraad oplossing in steriel dd water, (onmiddellijk voor te bereiden voor gebruik) </li><li> Gebruik CoCl 2 op de uiteindelijke concentratie van 100 urn in uw reguliere media voor celkweek tot hypoxie veroorzaken. </li><li> Voeg de CoCl 2 bevattende media aan je cellen en incubeer de culturen voor 24 uur in een conventionele incubator (37 ° C, 5% C0 2). </li></ol> De bovenstaande concentratie werkt voor de cellijnen die we hebben getest, maar elke cel lijn moet worden getest bij verschillende concentraties (om een ​​dosis-afhankelijke curve te stellen), evenals op de verschillende incubatie tijden om drugsgerelateerde toxiciteit te beperken en optimaliseren van de test. 2. Hypoxie geïnduceerd in Modulair Incubator Kamer <ol><li> Bereid ten minste twee identieke (twin cultuur) celculturen. Celculturen kunnen worden in vaten, borden of cultuur gerechten. Voor het openen van de kamer, opent u eerst de twee witte plastic klemmen op de buizen bevestigd aan de kamer (buizen gebruikt voor de injectie / zuiveren van het hypoxische gas) en open voorzichtig de stalen ring klem. </li><li> Laat de klem. Het deksel en bakken kunnen nu worden verwijderd. </li><li> Plaats de celcultuur in de hypoxische kamer. Ook plaats een petrischaal met steriele water in de kamer om een ​​adequate bevochtiging van de culturen te bieden. </li><li> Plaats de &quot;tweeling&quot; celkweek in normoxia als controle. </li><li> Zorg ervoor dat uw trays zijn beveiligd en niet bewegen, en sluit daarna de kamer met zijn deksel. De juiste positie van de stalen ring klem om de hermetische afsluiting van de kamer te verzekeren en te sluiten. </li><li> Voor het maken van hypoxie, bevestig de slang aan op een &quot;hypoxie tank&quot; met een 1% O 2 gasmengsel. Als u een flow meter is aangesloten op uw tank uw kamer zal direct worden aangesloten op het (gas tank-flow meter-kamer). We maken gebruik van een flow meter opgenomen in onze regulator. </li><li> Het is belangrijk om de meeste te verwijderen, zo niet alle aanwezige zuurstof in de kamer en in uw media, om dit te doen spoelen van de kamer door het openen van de gas tank met een debiet van 20 liter per minuut gedurende 7-10 minuten, daarna snel uit te schakelen van de gasstroom en volledig de kamer dicht bij het sluiten van beide witte klemmen. </li><li> Ga terug de kamer om een ​​conventionele incubator voor de gewenste periode. Als u grote culturen, laat de media in de cultuur om de-gas voor 1 - 2 uur en daarna spoelen te herhalen. De bovenstaande stappen zijn gebaseerd op de constructor aanbevelingen &#39;(Billups-Rothenberg, Inc). Zorg ervoor dat je benzinetank goed beveiligd te allen tijde. </li></ol> Opmerkingen: <ul><li> Om de O 2 in de media is geadviseerd om opnieuw gas de kamer een keer na een drie uur-te elimineren. </li><li> Voor incubatietijd langer dan 48 uur is het ook aangeraden om opnieuw gas de kamer. </li><li> Zuurstof sensoren (elektroden / manometer) die het mogelijk maken metingen van de O 2 in cellen / kamer zal zorgen voor een meer nauwkeurige meting van de intracellulaire / kamer O 2 bevatten). </li><li> Kweken van cellen die op verschillende tijdstippen moeite om een ​​tijd-curve te maken zou helpen bij het bepalen van de optimale tijd expressie van HIF-1α in uw cellen. </li></ul> 3. Evaluatie van Hypoxie <ol><li> HIF-1 α detectie door western blot. <ol><li> Opnieuw te openen uw kamer zoals beschreven in paragraaf 2.2 en onmiddellijk uw culturen op ijs. </li><li> Lyse hypoxie worden behandeld en niet-behandelde cellen met 5% SDS-oplossing in de platen vervolgens overbrengen naar de cel lyzate buizen, ultrasone trillingen, spindown de cel puin en het verzamelen van de supernatant. </li><li> Meet eiwitconcentratie met behulp van BCA kit, voor te bereiden monsters door het toevoegen van laadbuffer en de verwarming op 95 ° C gedurende 5 minuten. </li><li> Electrophorese eiwitten op een 8% SDS-polyacrylamide gel en transfer naar nitrocellulosemembranen. </li><li> Blokkeren de membraan in PBS dat 5% niet-vette droge melk en 0,1% Tween 20 bij kamertemperatuur gedurende 1 uur of bij 4 ° C gedurende de nacht op shaker. </li><li> Immunoblot &#39;s nachts met anti-HIF-1α primair monoklonaal antilichaam (1 / 600) in dezelfde buffer bij 4 ° C. </li><li> Was drie keer in PBS met 0,1% Tween 20 bij kamertemperatuur, 5min telkens en incubeer met HRP-secundair antilichaam (1 / 2000) in PBS + 0,1% Tween 20 gedurende 45 minuten. </li><li> Was drie keer in PBS met 0,1% Tween 20 bij kamertemperatuur, 5min / tijd. </li><li> Gebruik Pierce ECL-kit en X-ray film aan het eiwit band tonen </li></ol></li><li> Hypoxie reagerende element (HRE). HIF-1α regelt een groot aantal genen (dat wil zeggen VEGF, EPO) door binding aan een serie genaamd Hypoxie Regulering Element (HRE). Met behulp van HRE geassocieerd met een marker-gen it is het mogelijk om HIF-activiteit te detecteren 9. Voor het gebruik van deze methode, moet u eerst uw cellen transfecteren met een HRE-luciferase plasmide met behulp van een transfectie methode aangepast aan uw cellen. Zo gebruikten we Lipofectamine 2000. Cellen moeten worden getransfecteerd minstens 4 uur voor te worden geïncubeerd in hypoxie en normoxia. Deze keer kan het DNA van de cellen in te voeren, zodat deze eerste stap wordt niet beïnvloed door hypoxie. De luciferase signaal wordt gedetecteerd met behulp van een Luciferase Assay Kit. <ol><li> Incubeer uw HRE-getransfecteerde cellen in hypoxie in modulaire kamer of gebruik HRE-getransfecteerde cellen geïncubeerd met CoCl 2, omvatten een normoxia controle. </li><li> Na de gewenste incubatietijd, verwijder de groei medium van cellen. </li><li> Spoel cellen in PBS, verwijder de PBS. </li><li> Verdeel 400μl 1X lysis reagens in de cultuur schotel en schraap de bijgevoegde cellen, vervolgens overbrengen naar een micro-tube. </li><li> Pellet puin door korte centrifugeren. </li><li> Mix 20μl van cellysaten supernatant met 100μl van luciferase assay reagens en het meten van de luminescentie met behulp van een luminometer. </li></ol></li></ol> 4. Representatieve resultaten: In K562 cellen (humane leukemie cellijn) gecultiveerde twee dagen bij weinig zuurstof, in vergelijking met normoxia, hypoxie leidt tot een toename van HIF-1α eiwit dat werd gedetecteerd door western blot (figuur 1). Met behulp van HRE-luciferase aangepaste 293-cellen (embryonale nier cellijn) gekweekt in hypoxie, een significante toename van HIF-1α activiteit werd gedetecteerd in hypoxische cellen (figuur 2). <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Figuur 1"/> Figuur 1: Verhoging van HIF-1α in hypoxische cellen. K562 cellen werden in cultuur normoxia en hypoxie (hypoxische kamer) voor 48 uur en geanalyseerd door western blot met behulp van een antilichaam dat specifiek is voor HIF-1α. Een antilichaam dat specifiek is voor actine werd gebruikt voor het laden te controleren. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Figuur 2"/> Figuur 2: HIF-1α activiteit. HRE-luciferase aangepaste 293-cellen werden gekweekt in hypoxie en normoxia voor 48 uur en daarna gelyseerd met luciferase-signaal met behulp van een luciferase assay kit en een luminometer detecteren. De resultaten worden uitgedrukt als de relatieve luminescentie eenheid (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>

Geen belangenconflicten verklaard.

Celproliferatie en levensvatbaarheid in hypoxische omstandigheden nogal variëren, afhankelijk van de celtypen. Daarom moet u de cel of het nummer van de culturen platen je begint met ervoor zorgen dat je genoeg cellen / eiwitten hebben voor uw experimenten. De Cobalt Chloride methode heeft het voordeel om goedkoop en snel. Dit product bootst hypoxie door het induceren van HIF-1/3α maar kunnen ook andere genen reguleren, zorg ervoor dat het product wordt aangepast aan uw project door te kijken welke andere effecten onafhankelijk van elkaar kan hebben voor de functie en fenotype van uw specifieke cellen van het &quot;na te bootsen, hypoxische &quot;effect. Een ander geneesmiddel ook gebruikt voor de hypoxie na te bootsen is deferoxamine mesylaat (DFO, gebruikt bij 100 uM eindconcentratie). Het gebruik van deze drugs kan de experimentator met de cultuur plaat / schaal / fles vele malen te openen zonder dat de &quot;hypoxische toestand&quot;. Het niveau van zuurstof in het gasmengsel kan variëren afhankelijk van uw experimenten en celtypes als hypoxie waarden variëren afhankelijk van de weefsels en celtypes. Inderdaad, sommige cellen hypoxische in 5% O 2 andere hebben minder dan 1% O 2 wordt hypoxie. 5% CO 2 wordt toegevoegd aan het gasmengsel om de pH van de culturen te stabiliseren, is de rest van het gas meestal stikstof. Hypoxische kamers hebben het voordeel van het niet gebruiken van drugs die cel gedrag kan afwisselen onafhankelijk van de zuurstofspanning. Echter, niet alle types van experimenten worden gedaan als zuurstof weer in de kamer bij elke opening en dus vermindert hypoxie. Je zou kunnen overwegen deze factor in uw experimenten, hypoxie / re-oxygenatie is een specifieke aandoening die kan invloed hebben op bepaalde celtypen. Een alternatief is het gebruik van hypoxie werkstations (aangesloten op pre-mixed gas tanks of op gas mengsystemen) of groter hypoxie incubator (hypoxie verwerking van kamer met handschoenenkastje), waarmee de experimenten om media te veranderen en cellen te manipuleren in continue hypoxische omgeving. Diverse hypoxische kamers werden gecommercialiseerd de afgelopen tien jaar en dient te worden gekozen op basis van uw laboratorium gebruikte, projecten, ruimte, grootte en budget. Zorg altijd dat het goed gebruik en de conditie van uw Kamer om de juiste kweekomstandigheden te garanderen. In het algemeen bij het gebruik van een kamer, kan het niveau van hypoxie worden gemoduleerd door het selecteren van de verschillende gasmengsels (dwz 1%, 5% 10% zuurstof). Wat betreft de detectie van HIF-1α, is het belangrijk te weten dat sommige kanker cellijn te uiten HIF-1α in normoxia. Het is daarom van cruciaal belang voor een normoxia-control te gebruiken om de basale niveau van HIF in deze cellijnen te bepalen. HIF-1α kunnen ook aanwezig in normoxia in niet-maligne cellen worden na cel stimulatie of stress. Dit kan ook optreden als deze cellen worden uitgehongerd, zorg ervoor dat je celkweken goed voeden en onderhouden.

<table border="1"><tbody><tr><th> Naam van het serum </th><th> Vennootschap </th><th> Catalogus nummer </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Kobalt (II) Chloride </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> Incubator Kamer </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> HRE-Luciferase plasmide </td><td> Adgene </td><td> Plasmide 26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

Hypoxie is gedefinieerd als de vermindering of gebrek aan zuurstof in organen, weefsels en cellen. Deze daling van zuurstof spanning kan te wijten zijn aan een verminderde aanvoer van zuurstof (oorzaken zijn onvoldoende bloedvat netwerk, defecte bloedvat, en bloedarmoede) of een verhoogd verbruik van zuurstof ten opzichte van het aanbod (veroorzaakt door een plotselinge hogere celproliferatie tarief) . Hypoxie kan worden fysiologische of pathologische, zoals in vaste kanker 1-3, reumatoïde artritis, atherosclerose etc ... Elke weefsels en cellen hebben een andere staat aan te passen aan deze nieuwe voorwaarde. Tijdens de hypoxie, is hypoxia-induceerbare factor-alfa (HIF), die stabiel en regelt verschillende genen, zoals die betrokken zijn bij angiogenese of transport van zuurstof 4. De stabilisering van dit eiwit is een kenmerk van hypoxie, dus het detecteren van HIF wordt routinematig gebruikt om te screenen op hypoxie 5-7. In dit artikel stellen we twee eenvoudige methoden om hypoxie te induceren in zoogdiercellen culturen en eenvoudige tests om de hypoxische status van deze cellen te evalueren.

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

Hier hebben we voorstellen eenvoudige methoden om hypoxie te induceren in celkweken en eenvoudige tests om de hypoxische status van de culturen te evalueren.

Inductie en testen van Hypoxie in celkweek

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 ...

induction-and-testing-of-hypoxia-in-cell-culture

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Biology

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.

Cultuur van Mouse Neurale Stem Cell Voorlopers

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

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.

Inductie en Monitoring van Adoptieve vertraagd-type overgevoeligheid bij ratten

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.

Efficiënte Afleiding van de Mens Cardiac precursoren en cardiomyocyten uit pluripotente menselijke embryonale stamcellen met op kleine moleculen Inductie

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%.

Isolatie en Primaire Cultuur van Rat levercellen

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.

De Slice kweekmethode voor Na Ontwikkeling van Tooth Germs In explantaatkweek

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;

Isolatie, Cultuur en Transplantatie van Muscle satelliet-cellen

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.

Analyse van Global RNA synthese in de Single Cell Level volgende Hypoxie

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.

De c-FOS Protein Immunohistologische Detection: Een handig hulpmiddel als een marker van Central Pathways betrokken bij specifieke fysiologische reacties<em&gt; In Vivo</em&gt; en<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.

Detectie van RNA-bindende eiwitten door<em&gt; In Vitro</em&gt; RNA Pull-down in Adipocyte Cultuur

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 ...

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.