Die Autoren möchten die Creative Services Team an der University of Missouri Academic Support Center, die dieses Video produziert danken. Diese Arbeit wurde von der Eunice Kennedy Shriver National Institute of Child Health &amp; Human Development des National Institutes of Health, Grantnummer HD055231 unterstützt

Ein. Dissection der Maus Plazenta <ol><li> Richten Sie Fügepaaren von Mäusen. </li><li> An jedem der folgenden Tage, für die Anwesenheit eines copulator Stecker überprüfen. Der Tag, an dem ein Stecker erkannt wird als 0,5 Tage post-coitus (DPC) 7. </li><li> Plazenten können zu einem gewünschten Stadium der Schwangerschaft gesammelt werden, mit einem sauberen Dissektion der Plazenta möglichen Beginn mit der Entwicklung des reifen Plazenta auf d 10.5. Hier zeigen wir, 14,5 dpc. Bei den angegebenen Laufwerken wird 40-60 Plazenten auf 10,5 dpc und 10-20 Plazenten auf 14,5 dpc ergeben eine deutlich sichtbare Trophoblasten Zellschicht. </li><li> Euthanize die schwangere Maus, und entfernen Sie die Gebärmutter, Schneiden an der Zervix, und entlang der mesometrium. </li><li> Mit einer scharfen Schere zwischen jedem conceptus (Abbildung 1A) geschnitten. </li><li> Mit zwei Paar Zangen, fassen Sie die Gebärmutterwand an der Schnittkante und auseinanderziehen, das Entfernen des Myometrium (Abbildung 1B)<html> . Ein Paar gezackte, mäßig fein (gebogen oder gerade) Pinzette plus ein Paar sehr feinen Pinzette (# 5, zum Beispiel) wird empfohlen. </li><li> Gegenüber der Plazenta, öffnen Sie das decidua mit einer Pinzette oder Schere, Aussetzen Plazenta. Greifen Gebärmuttergewebe mit einer Pinzette Paar, schieben weitere Pinzette unterhalb der Plazenta zu trennen (Abbildung 1C). Falls erforderlich, separate Fötus und fetalen Membranen aus Plazenta. Es ist nicht möglich, alle dezidualen Gewebe durch Dissektion ohne auch verlieren Plazentagewebe entfernen, aber zusätzliche dezidualen Gewebe (blass) können von der Oberfläche der Plazenta durch leichtes Ablösen mit einer Pinzette entfernt werden. Wir werden auf der Percoll-Gradienten zur weiteren Reinigung angewiesen. </li><li> Platzieren Sie jede seziert Plazenta in 25 ml eiskaltem Dissoziationspuffer in einem 50 ml konischen Röhrchen, bis alle Sektionen vollständig sind. In späteren Stadien, grob zerkleinern Plazentagewebe mit einer Schere oder Rasierklinge, bevor sie in Dissoziationspuffer übertragen. </li></ol>tle &quot;&gt; 2. Isolierung von Trophoblastzellen <ol><li> Locker cap konischen Rohr und in einem 37 ° C Bad für 45-60 Minuten. Mischen Sie die Dissoziation Lösung kräftig durch Pipettieren ca. alle 10 min. Die gesamte Dauer der Verdauung sollten engmaschig überwacht werden und wird wahrscheinlich verlangen einige trial and error, um eine optimale Erholung der Zellen zu erreichen. Gewebe sind in der Lage, durch einen 10 ml-Pipette übergeben werden, aber sichtbar Gewebestücke bleibt. Overdigestion wird in armen Zellüberleben und Platierungseffizienz führen. Optimal Zellüberleben und Platierungseffizienz wird auftreten, wenn Klumpen von wenigen Zellen vorhanden sind, anstatt wenn vollständige Dissoziation erreicht. Umgekehrt wird underdigestion reduziert Reinheit nach Percoll Trennung führen. Wenn die richtige Verdauung Punkt erreicht ist, an Ort Röhrchen auf Eis zu stoppen Kollagenase-Aktivität. </li><li> Übergeben Sie die Lösung durch eine Zelle Sieb, um unverdaute Materialien zu entfernen, und zentrifugieren @ 500 xg für 5 Minuten. </li><li> Zu waschen, zu entfernenÜberstand und resuspendieren Zellen in 10 ml Waschlösung. Zentrifuge @ 500 xg für 5 Minuten. </li><li> Während der Zentrifugation, bereiten Percoll Lösung durch Mischen 9,6 ml Percoll, 13,4 ml Waschlösung und 1,1 ml 10x Medium 199 in einem 50-ml-Röhrchen für geeignete Hochgeschwindigkeitszentrifugation. </li><li> Überstand entfernen und Zellpellet in 2 ml Waschlösung. </li><li> Hinzufügen von Zellen zur Lösung Percoll und zentrifugieren @ 30.000 xg für 30 Minuten bei 4 ° C. </li><li> Röhrchen aus Zentrifuge, ohne die Steigung. Um den Gradienten, Platz Rohr vor einer hellen Lichtquelle zu visualisieren. Suchen Sie den Trophoblasten Band (Abbildung 2), und saugen mit einer Pipette, kümmert sich nicht um die roten Blutkörperchen Band unterhalb stören. Volume des Trophoblasten Band 5-8 ml betragen. </li><li> Übertragen Trophoblastenzellen mit einem 50 ml konischen Röhrchen. Hinzufügen Waschlösung auf ein Gesamtvolumen von 45ml. </li><li> Zentrifuge @ 500 xg für 5 Minuten. Überstand entfernen, und resuspendieren in Kultur medium (NCTC-135). Zellen können auf dünnschichtigen Matrigel oder Gewebekultur-Plastik bei 37 ° C und 5% CO 2 kultiviert werden. Am Tag 10,5 ergibt jeweils Plazenta rund 50.000 lebenden Zellen, von denen 80-90% Cytokeratin positive sind. </li></ol> 3. Trophoblastinvasion Assay <ol><li> 1-2 ml Kulturmedium (NCTC-135) zu jedem Matrigel Kammer und bei 37 °, 5% CO 2 für 30-90 min. </li><li> Entfernen Medium und 1,5 ml x 10 5 frisch isolierten Zellen in 2ml mittleren bis oberen Kammer. 1 ml Medium unteren Kammer unteren Membran bedecken. </li><li> Kultur Zellen in Einsätze für 18-24 Stunden. </li><li> Scrape Zellen und Matrigel aus dem Einsatz mit einem Schaum-Tupfer. </li><li> Spülen der Oberseite und der Unterseite der Kammer in 2 ml PBS. </li><li> Fix für 5 min in 1 ml 4% Paraformaldehyd in PBS. </li><li> Stain, indem untere Kammer mit 400 ul DAPI (300nm) und Inkubation für 30 min. </li><li> Dreimal kurz in PBS. </li><li&gt; entfernen Membranen aus Einsätzen mit einem scharfen Skalpell. </li><li> Fügen Sie einen 10 ul Tropfen Eindeckmedium (50% Glycerin in PBS) zu rutschen. Mit einer Pinzette, lag Membran auf Drop. Hinzufügen 10 ul Tropfen des Mediums, dann vorsichtig Deckglas. </li><li> Photograph und Anzahl Zellen. Seien Sie sicher zu orientieren die Membran entsprechend (Zell-Seite nach oben oder Zell-Seite nach unten) für das Mikroskop verwendet wird. Image J Software (National Institutes of Health) eine manuelle Zählung Funktion, die für diesen Zweck verwendet werden können, ebenso wie Adobe Photoshop oder einem manuellen Zähler. </li></ol> 4. Repräsentative Ergebnisse <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Dissection der Maus Plazenta. (A) Der Uterus wird durch Schneiden entlang den Eileiter (gelb), entlang der Oberfläche mesometrial entfernt und an der Zervix (unten). Individuelle conceptuses kann dann durch Schneiden mit einer Schere getrennt dargestellt werden. (B) Der Uterus<html> Wand durch Anordnen zweier Pinzetten im Schnitt uterinen Oberfläche und Reißen entlang der Anti-mesometrial Seite, in der Nähe des Fötus und weg von der Plazenta entnommen. (C) Die Plazenta wird dann durch Greifen mit einer Zange und einem anderen zwischen der gleitenden Plazenta und Gebärmuttergewebe isoliert. Nabelschnur und anderen extraembryonalen Membranen können dann aus der Plazenta abgetrennt werden. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Percoll Trennung von Trophoblastzellen. Nach dem Zentrifugieren wird drei großen Bands zu sehen sein, wie in der Karikatur auf der linken Seite gezeigt, und fotografieren rechts. Die obere Bande enthält Zelltrümmern und Fibroblasten und erst am deutlichsten. Eine enge (rot) Band nahe der Unterseite mit roten Blutkörperchen sichtbar sein wird knapp unterhalb der diffuse Trophoblasten Band. Zellaggregate wird wahrscheinlich in den Trophoblasten Band gesehen, wie in den beiden direkt links des Pfeils auf dem Foto gezeigt. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Invasive Trophoblastenzellen (weißer Pfeil) wird durch die Schicht und den Matrigel-Membran Poren passieren, die auf der unteren Oberfläche der Membran. Die Poren sind sichtbar (schwarz Pfeilspitze). Die Anzahl der Zellen auf der gesamten Membranoberfläche oder einer repräsentativen Bereich gezählt werden. Ein Graustufenbild fluoreszierender DAPI Kernfärbung wurde umgedreht, um Visualisierung von Zellen zu verbessern. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Trophoblastzellen können identifiziert werden, und die Reinheit des isolierten Population beurteilt, durch Immunfärbung für Cytokeratin 7 (Mitte, Magenta). Die Zellen werden mit DAPI (oben, blau) gegengefärbt. Zusammengefügte Bild, unten.

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

Wir haben nichts zu offenbaren.

In diesem Video zeigen wir die Isolierung von Trophoblastzellen aus der Maus Plazenta. Wir zeigen dann eine in vitro-Assay für Trophoblastinvasion. Mit diesem Verfahren wird eine weitgehend, aber nicht vollständig, können reine Population von Trophoblasten Zellen gewonnen werden. Wenn weitere Reinheit erforderlich ist, könnte Magnetkügelchen Trennung oder Durchflusszytometrie durchgeführt werden, wie es für primäre humane Trophoblastenzellen beschrieben worden. Die richtige Länge der Kollagenase Dissoziationssc...

Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
<table border='1'>
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>50ml</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>2.383 g</td>
 <td>0.02M</td>
 </tr>
 <tr>
 <td>sodium bicarbonate</td>
 <td>0.42 g</td>
 <td>0.01M</td>
 </tr>
 <tr>
 <td>Penicllin-Streptomycin</td>
 <td>5 mL</td>
 <td>100 U- 100 &mu;g /ml</td>
 </tr>
 <tr>
 <td>Sterile dH20</td>
 <td>to 500 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Dissociation solution (make fresh and filter sterilize)
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Wash Solution</td>
 <td>100 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>1 vial (2000 U)</td>
 <td>20 U/mL</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>100 mg</td>
 <td>125 digestion units/mL</td>
 </tr>
 </tbody>
</table>


<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C9891</td>
 <td>This is Clostridium collagenase.
Bovine pancreatic collagenase would likely work as well</td>
 </tr>
 <tr>
 <td>NCTC-135</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>Add FBS to 10%</td>
 </tr>
 <tr>
 <td>Matrigel invasion chambers</td>
 <td>BD Biosciences</td>
 <td>354481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>Sigma</td>
 <td>M9163</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m Cell strainer</td>
 <td>Fisher</td>
 <td>22363549</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibody to cytokeratin 7</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Used at 1:100 dilution</td>
 </tr>
 </tbody>
</table>

Equipment Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.

isolation of primary mouse trophoblast cells and trophoblast invasion assay

Die Plazenta ist verantwortlich für den Transport von Nährstoffen, Gasen und Wachstumsfaktoren für den Fötus, sowie die Beseitigung von Abfällen. So haben Mängel in Plazentaentwicklung wichtige Konsequenzen für den Fötus und Mutter, und sind eine der Hauptursachen für embryonale Letalität. Der wesentliche Zelltyp des fetalen Teils der Plazenta ist die Trophoblasten. Primäre Maus plazentalen Trophoblastenzellen sind ein nützliches Werkzeug für die Untersuchung normalen und abnormalen Plazentaentwicklung, und anders als Zelllinien, können isoliert und verwendet werden, um Trophoblasten in spezifischen Stadien der Schwangerschaft zu studieren. Zusätzlich kann Primärkulturen von Trophoblasten aus transgenen Mäusen verwendet, um die Rolle von bestimmter Gene in Plazenta-Zellen zu studieren. Die hier vorgestellten Protokoll basiert auf der Beschreibung von Thordarson et al. 1, bei dem eine Percoll Gradienten verwendet werden, um ein relativ reines Trophoblasten Zellpopulation aus isolierten Maus Plazenten erhalten wird. Es ist ähnlich wie die weiter verbreitet verwendetd Methoden für menschliche Trophoblasten Zellisolation 2-3. Reinheit kann durch immuncytochemische Anfärben der isolierten Zellen für Cytokeratin 7 4 bewertet werden. Hier werden die isolierten Zellen dann unter Verwendung eines Assays, um Matrigel Invasion Trophoblasten Invasivität in vitro 5-6 beurteilen. Die befallenen Zellen werden durch Immunzytochemie und gebeizt zum Zählen analysiert.

Watch this Scientific Journal Video about Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay at JoVE.com

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

In diesem Protokoll beschreiben wir die Zerlegung der Plazenten von der Maus auf die Schwangerschaft d10.5, durch Isolation von Trophoblastzellen mit einem Percoll-Gradienten folgen. Wir demonstrieren dann Verwendung der isolierten Zellen in einem Matrigel Invasionsassay.

Isolation of Primary Maus Trophoblastzellen und Trophoblastinvasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.6K Aufrufe.  University of Missouri. In diesem Protokoll beschreiben wir die Zerlegung der Plazenten von der Maus auf die Schwangerschaft d10.5, durch Isolation von Trophoblastzellen mit einem Percoll-Gradienten folgen. Wir demonstrieren dann Verwendung der isolierten Zellen in einem Matrigel Invasionsassay.

Serie: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Ableitung der Maus Trophoblast Stammzellen aus Blastozysten

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Forschung

Biologie

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%.

Isolierung und Primärkultur von Ratten Leberzellen

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

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolierung von Immunzellen aus Primärtumoren

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolierung und Kultivierung von primären Maus Azinuszellen des Pankreas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust 3D DNA FISH Mit Direkt markierten Sonden

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Isolierung von Primär Myofibroblasten aus Maus und Mensch Colon Tissue

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Isolierung, Charakterisierung und hohem Durchsatz extrazelluläre Flux-Analyse der Maus primäre renale tubuläre Epithelzellen

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

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.

Einzelzellsammlung von Trophoblastenzellen im Peri-Implantationsstadium menschliche Embryonen

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