Gli autori desiderano ringraziare il team Creative Services presso l&#39;Università del Missouri Centro di supporto accademico che ha prodotto questo video. Questo lavoro è stato sostenuto dal Kennedy Eunice Shriver National Institute of Child Development &amp; Salute umana del National Institutes of Health, un numero di concessione HD055231

1. Dissezione della placenta del mouse <ol><li> Impostare coppie di accoppiamento di topi. </li><li> In ciascuno dei giorni seguenti, verificare la presenza di una spina copulatore. Il giorno in cui viene rilevato un tappo è considerato 0,5 giorni post-coito (DPC) 7. </li><li> Placente possono essere raccolte nella fase desiderata della gravidanza, con una dissezione pulita dell&#39;inizio placenta possibile con lo sviluppo della placenta maturo d 10,5. In questo studio dimostriamo 14,5 dpc. Ai volumi dati, 40-60 placente da 10,5 dpc, e 10-20 su placente 14,5 dpc produrrà un ben visibile strato di cellule del trofoblasto. </li><li> Eutanasia il mouse in stato di gravidanza, e togliere l&#39;utero, il taglio al collo dell&#39;utero, e lungo il mesometrium. </li><li> Utilizzo di forbici affilate, tagliate tra ogni concepimento (Figura 1A). </li><li> Utilizzando due coppie di pinze, afferrare la parete uterina al bordo di taglio, e separare, rimuovendo il miometrio (Figura 1B)<html> . Un paio seghettato, pinze moderatamente fini (curvo o diritto) più una coppia pinza molto fini (# 5, per esempio) è raccomandato. </li><li> Di fronte la placenta, aprire la decidua con pinze o forbici, esponendo placenta. Cogliere tessuto uterino con una coppia pinze, far scorrere un altro pinza sotto la placenta per separare (Figura 1C). Se necessario, il feto separato e membrane fetali di placenta. Non è possibile rimuovere tutti i tessuti decidual da dissezione anche senza perdere tessuto placentare, ma ulteriore tessuto decidual (pale) può essere rimosso dalla superficie della placenta peeling delicatamente con una pinza. Contiamo sul gradiente Percoll per ulteriore purificazione. </li><li> Mettete ogni placenta sezionato in 25 ml di tampone di ghiaccio freddo dissociazione in una provetta da 50 ml fino a quando tutte le dissezioni sono completi. Nelle fasi successive, tritare grossolanamente tessuto placentare con le forbici o lama di rasoio prima del trasferimento nel buffer di dissociazione. </li></ol>tle &quot;&gt; 2. Isolamento di cellule del trofoblasto <ol><li> Liberamente provetta con tappo conico e posto in un bagno a 37 ° C per 45-60 minuti. Miscelare la soluzione dissociazione vigorosamente pipettando circa ogni 10 min. Il tempo di digestione totale dovrebbe essere attentamente monitorati e probabilmente richiederà un po &#39;di tentativi ed errori per ottenere un recupero ottimale delle cellule. Tessuti dovrebbe essere in grado di essere fatto passare attraverso una pipetta 10 ml, ma visibili pezzi di tessuto rimarranno. Overdigestion si tradurrà in scarsa sopravvivenza delle cellule e l&#39;efficienza placcatura. Sopravvivenza cellulare ottimale e l&#39;efficienza placcatura si verifica quando grumi di poche cellule sono presenti, piuttosto che quando completa dissociazione è raggiunto. Al contrario, underdigestion porterà a purezza ridotta in seguito alla separazione Percoll. Quando il punto di digestione è stato raggiunto, il tubo posto in ghiaccio per fermare le attività collagenasi. </li><li> Filtrare la soluzione attraverso un colino cellula per rimuovere i materiali non digeriti, e centrifugare a 500 xg per 5 minuti. </li><li> Per il lavaggio, rimuoverecellule surnatante e risospendere in 10 ml di soluzione di lavaggio. Centrifugare a 500 xg per 5 minuti. </li><li> Durante la centrifugazione, preparare la soluzione di Percoll miscelando 9,6 ml di Percoll, 13,4 ml di soluzione di lavaggio e di 1,1 ml di mezzo 10x 199 in un tubo da 50 ml appropriato per centrifugazione ad alta velocità. </li><li> Rimuovere il surnatante e risospendere il pellet di cellule in 2 ml di soluzione di lavaggio. </li><li> Aggiungere celle Percoll soluzione e centrifugare @ 30.000 xg per 30 minuti a 4 ° C. </li><li> Rimuovere il tubo da centrifuga senza disturbare il gradiente. Per visualizzare il, tubo posto gradiente di fronte a una fonte di luce intensa. Individuare la banda trofoblasto (Figura 2), e aspirare con una pipetta, facendo attenzione a non disturbare la banda rossa globuli sotto. Volume della band trofoblasto sarà 5-8 ml. </li><li> Trasferimento cellule del trofoblasto in una provetta da 50 ml. Aggiungere la soluzione di lavaggio per un volume totale di 45 ml. </li><li> Centrifugare a 500 xg per 5 minuti. Rimuovere il surnatante e risospendere nella cultura medium (NCTC-135). Cellule possono essere coltivate su strato sottile o matrigel coltura tissutale di plastica a 37 ° C e 5% di CO 2. Al giorno 10.5, ogni placenta produce circa 50.000 cellule vive, di cui il 80-90% sono citocheratina positivi. </li></ol> 3. Trofoblasto invasione Assay <ol><li> Aggiungere 1-2 ml di terreno di coltura (NCTC-135) a ciascuna camera di matrigel e incubare a 37 °, 5% CO2 per 30-90 min. </li><li> Estrarre il supporto e aggiungere 1,5 ml x 10 5 cellule isolate di fresco in mezzo 2ml di camera superiore. Aggiungere 1 ml di mezzo di camera inferiore per coprire superficie della membrana inferiore. </li><li> Cellule in coltura per 18-24 ore inserti. </li><li> Cellule raschiare e Matrigel da l&#39;inserto utilizzando un tampone di schiuma. </li><li> Risciacquare parte superiore e inferiore della camera in 2 ml di PBS. </li><li> Fix per 5 min in 1 ml di paraformaldeide 4% in PBS. </li><li> Stain compilando Camera bassa con 400 microlitri DAPI (300 nM) e incubare per 30 min. </li><li> Lavare tre volte brevemente in PBS. </li><li&gt; Rimuovi membrane da inserti con una lama affilata bisturi. </li><li> Aggiungere una goccia 10 ml di mezzo di montaggio (50% glicerolo in PBS) a scivolare. Utilizzando pinze, laici membrana su goccia. Aggiungi un&#39;altra goccia 10 ml di mezzo, quindi coprioggetti con attenzione. </li><li> Fotografie e il numero di cellule. Accertarsi di orientare la membrana in modo appropriato (cellulare rivolta verso l&#39;alto o cellulare rivolto verso il basso) per il microscopio utilizzato. Software Image J (National Institutes of Health) include una funzione di conteggio manuale che può essere utilizzato per questo scopo, come può Adobe Photoshop o un contatore manuale. </li></ol> 4. Risultati rappresentativi <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Figura 1" /> Figura 1. Dissezione della placenta mouse. (A) L&#39;utero viene rimosso tagliando lungo le ovidotti (giallo), lungo la superficie mesometrial, e nella cervice (in basso). Conceptuses individuali possono essere separati mediante taglio con le forbici come mostrato. (B) Il uterina<html> parete viene rimossa inserendo due pinze nella superficie di taglio uterina, e strappando lungo l&#39;anti-mesometrial lato, vicino al feto e dalla placenta. (C) La placenta viene poi isolato afferrando con una pinza e scorrevole altro tra la placenta e tessuto uterino. Cordone ombelicale e altre membrane extraembrionali può quindi essere separata dalla placenta. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Figura 2" /> Figura 2. Percoll separazione di cellule trofoblastiche. Dopo la centrifugazione, tre fasce principali sarà visibile, come mostrato nella vignetta a sinistra, e la fotografia a destra. La fascia superiore contiene residui cellulari e fibroblasti e sarà più evidente. A stretto (rossa) banda in prossimità del fondo contenente globuli rossi sarà visibile appena sotto la fascia trofoblasto diffuso. Aggregati cellulari probabilmente visto nella banda trofoblasto, come mostrato nelle due appena a sinistra della freccia nella fotografia. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Figura 3" /> Figura 3. Trofoblasti invasive (freccia bianca) passerà attraverso lo strato di matrigel e dei pori della membrana, che appare sulla superficie inferiore della membrana. I pori sono visibili anche (nero punta di freccia). Il numero di cellule sulla superficie della membrana intera o una zona campione rappresentativo può essere contato. Una scala di grigi di colorazione fluorescente DAPI nucleare è stato invertito per migliorare la visualizzazione delle cellule. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Figura 4" /> Cellule del trofoblasto Figura 4. Possono essere identificati, e la purezza della popolazione isolata valutato, mediante immunocolorazione per la citocheratina 7 (centro, magenta). Cellule sono colorate con DAPI (alto, blu). Uniti immagine, in basso.

<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>

Non abbiamo nulla da rivelare.

In questo video, dimostriamo l&#39;isolamento di cellule del trofoblasto della placenta mouse. Abbiamo poi mostrano un saggio in vitro per l&#39;invasione del trofoblasto. Utilizzando questa procedura, una gran parte, ma non completamente, popolazione pura di cellule trofoblastiche può essere ottenuta. Se la purezza è necessario un ulteriore, separazione magnetica tallone o citometria di flusso potrebbe essere eseguita, come è stato descritto per primarie cellule trofoblastiche umane. La corretta lunghezza del passo ...

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

La placenta è responsabile per il trasporto di sostanze nutritive, gas e fattori di crescita per il feto, nonché l&#39;eliminazione dei rifiuti. Così, difetti di sviluppo della placenta hanno importanti conseguenze per il feto e la madre, e sono una delle principali cause di mortalità embrionale. Il tipo cellulare principale della porzione fetale della placenta è trofoblasto. Primarie trofoblasti placentari topo sono un utile strumento per studiare normale e anormale sviluppo placentare, ea differenza linee cellulari, possono essere isolati e utilizzati per studiare trofoblasto in determinate fasi della gravidanza. Inoltre, colture primarie di trofoblasto di topi transgenici possono essere utilizzati per studiare il ruolo di particolari geni in cellule placentari. Il protocollo qui presentato è basato sulla descrizione da Thordarson et al. 1, in cui viene utilizzato un gradiente Percoll per ottenere una popolazione di cellule relativamente puro trofoblasto da placente mouse isolato. È simile a utilizzare il più ampiamentemetodi per l&#39;uomo d 2-3 isolamento cellulare trofoblasto. Purezza può essere valutata mediante colorazione immunocitochimica delle cellule isolate per citocheratina 7 4. Qui, le cellule isolate vengono quindi analizzati utilizzando un saggio di invasione matrigel a valutare invasività trofoblasto in vitro 5-6. Le cellule invase vengono analizzati mediante immunocitochimica e tinto per il conteggio.

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 questo protocollo, si descrive la dissezione placentae dal mouse sulla gravidanza D10.5, seguito da isolamento di cellule trofoblastiche mediante gradiente di Percoll. Abbiamo poi dimostrare l&#39;utilizzo delle cellule isolate in un saggio di invasione Matrigel.

Isolamento di cellule primarie trofoblasto mouse e Invasion Assay trofoblasto

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 ...

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Research

JoVE Journal

Biology

23.6K Views.  University of Missouri. In questo protocollo, si descrive la dissezione placentae dal mouse sulla gravidanza D10.5, seguito da isolamento di cellule trofoblastiche mediante gradiente di Percoll. Abbiamo poi dimostrare l'utilizzo delle cellule isolate in un saggio di invasione Matrigel.

Video: 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.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

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

Ricerca

Biologia

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 ...

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.

Isolamento di cellule immunitarie di tumori primari

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.

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

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.

Robusto FISH 3D DNA con sonde marcate direttamente

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.

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

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.

Isolamento e colture primarie di cellule di topo aortica endoteliali

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.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

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.

Isolamento, caratterizzazione e Throughput elevato flusso extracellulare analisi di cellule epiteliali tubolari renali primari del Mouse

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.

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

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