We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Levental, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Carson, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+characterization+of+ex+vivo+prostate+tissue+via+spherical+indentation+in+the+clinic.">Material characterization of ex vivo prostate tissue via spherical indentation in the clinic.</a> Med Eng Phys. 33 (3), 302-309 (2011).</li><li>Hoyt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+elasticity+properties+as+biomarkers+for+prostate+cancer.">Tissue elasticity properties as biomarkers for prostate cancer.</a> Cancer Biomark. 4 (4-5), 213-225 (2008).</li><li>Krouskop, T. A., Wheeler, T. M., Kallel, F., Garra, B. 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The authors have nothing to disclose.

Un protocollo per la generazione di acini ghiandolari 3D da MECs a puro gel rbm 6 è stato modificato in uno studio precedente con l&#39;aggiunta di 4 mg tipo / ml collagene alla matrice MLF. L&#39;aggiunta di collagene provocato il modulo elastico del gel meccanismi crescenti da 175 ± 37 al 1589 ± 380 Pascal. Questo aumento 9,1 volte della rigidità modulata la crescita, la sopravvivenza, la migrazione e la differenziazione delle MECs 21. Il protocollo è stato modificato nuovamente includendo una fase di trattamento con D - (-) - ribosio promuovere reticolazione non enzimatica del collagene di tipo I che era stato aggiunto al gel MLF. Il risultante aumento di 15 volte della rigidità è stato trovato a collaborare con la trasformazione oncogenica di MECs per promuovere il loro comportamento invasivo 22. L&#39;approccio sperimentale di tipo aggiunta di collagene di gel RBM facilita l&#39;interazione diretta di MECs con fibre di collagene, che si verifica solo nei tessuti umani dopo la barriera fisica tra lo stromaed epitelio fornite dal BM subisce degradazione proteolitica. Generando acini ghiandolari 3D da PEC in gel puro RBM pre-trattati con GLA, l&#39;attuale protocollo apre la strada per studiare come BM rigidità di per sé in grado di attivare il loro comportamento invasivo (Figura 3). I livelli di BM rigidità indotte in questo protocollo hanno rilevanza fisiologica. L&#39;incubazione con 50 mM GLA per 6 ore e 14 ore, rispettivamente aumentato i moduli elastici del gel puro meccanismi a 175 ± 90 e 322 ± 160 rispetto a 122 ± 55 Pascal in gel MLF trattati con PBS (Tabella 1). Questo aumento 1,7-3,2 volte in RBM rigidità ricapitola il 2,5 a 3,4 volte aumento della rigidità osservato in maligno della prostata rispetto al normale o tessuto di BPH 23-26. Come indicato in una recente pubblicazione 13 le variazioni morfologiche indotte da un accumulo di AGE e RBM rigidità PEC acini può essere quantificato per un cambiamento statisticamente significativo da arounded a forma poligonale, diminuzione del lume / superficie totale acinare, e le cellule sporgenti che migrano dal acina nel ricco-AGE RBM (Figura 3). Immunoblotting può anche essere utilizzato per valutare marcatori di EMT (ad esempio perdita di E-caderina 13) e il comportamento contrattili (es fosforilata leggera della miosina catena 2, pMLC2 13) in PEC coltivati ​​in normale rispetto rigida MLF (Figura 3). Ulteriore valutazione usando immunofluorescenza e microscopia confocale può essere applicata per visualizzare il BM (es laminina, collagene IV e accumulo di AGE 13), cellulare polarità apicale-to-basale (es localizzazione apicale EEA1: primi endosomal antigene 1 e GM130: 130 kDa marcatore cis-Golgi 13) e modelli cellulari di molecole di adesione (es localizzazione e-caderina per giunzioni cellula-cellula 13) (Figura 3). <img alt = &quot;Figura 3&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig3.jpg&quot; /&gt; Figura 3:. Panoramica dei diversi protocolli qui presentati Il diagramma illustra come preparare e irrigidire la membrana basale ricostituita (RBM) con glicolaldeide (reazione di Maillard), come seme le cellule al rigida RBM, come analizzare la rigida RBM ( entità della reazione di Maillard) e le procedure che possono essere utilizzate per analizzare i cambiamenti cellulari e molecolari indotte da ricchi-AGE RBM. AGE, prodotti finali della glicazione avanzati; BM, membrana basale; DAPI, 4 &#39;, 6-diamidino-2-phenylindole; EEA1, presto antigene endosomal 1; GAPDH, gliceraldeide-3-fosfato deidrogenasi; GLA, glicolaldeide; GEE, glicina estere etilico; GM130, 130 kDa marcatori cis-Golgi; p-MLC2 (Thr18 / Ser19), miosina luce catena 2 fosforilata in siti treonina 18 e serina 19; RBM, membrana basale ricostituita; SDS-PAGE, sodio dodecil solfato gel di poliacrilammide. Per la barra RWPE1 acini Scala = 10 micron; per cellule tumorali PC3bar sferoidi Scala = 100 micron. Questa cifra è stata modificata dal riferimento 13. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> saranno necessari passaggi di risoluzione dei problemi se D - (-) - ribosio viene scelto come agente reticolante per RBM. Durante lo sviluppo del protocollo è stato trovato che il trattamento con 1 M D - (-) - ribosio per 72 ore, come precedentemente descritto per i gel RBM / collagene 22, portato alla disidratazione e ritiro del gel MLF. La valutazione delle concentrazioni inferiori di D - (-) - ribosio e tempi di trattamento più brevi può contribuire a superare questa limitazione. Un potenziale limitazione future applicazioni del protocollo potrebbe essere incontrata in cui si desiderano livelli più alti di RBM rigidità. Se i tempi di incubazione più lunghi e concentrazioni più elevate di GLA vengono utilizzati per indurre livelli elevati di MLF gel rigidità sarà necessario<html> valutare se tali condizioni di trattamento hanno un impatto sulla sopravvivenza e la proliferazione cellulare, come descritto in precedenza 13. Va inoltre notato che l&#39;incubazione di RWPE-1 le cellule con siero induce una transizione EMT-like fenotipica e l&#39;esposizione a siero o materiali contenenti siero deve essere evitato. Ad esempio, se gli esperimenti riguardano la transfezione di short interfering RNA oligonucleotidi (siRNA), la procedura deve essere ottimizzato usando le cellule RWPE-1 coltivate in KSFM, senza passare le cellule su un supporto a basso siero trasfezione. Questo inconveniente potrebbe compromettere il livello di silenziamento genico ottenuto utilizzando siRNA transitorio si avvicina nel modello. Per alcuni bersagli proteici che sarebbe stato consigliato di utilizzare vettori shRNA inducibili per sintonizzabile silenziamento genico e la diminuzione desiderata in livelli di proteine. Adattamenti che incorporano reticolazione enzimatica da cellule stromali o cellule tumorali lysyl associato ossidasi (LOX) 17 potrebbe anche essere incorporati in modelli futuri. jove_content &quot;&gt; Questo protocollo faciliterà il futuro studio dei meccanismi pro-invasive attivati ​​da età-dipendente BM rigidità PEC (RWPE-1, BPH-1) e la valutazione degli obiettivi anti-metastatici in PTC invasive (PC3). Dato che BPH è considerato come un disordine metabolico 27, questo protocollo spiana anche la strada verso la nostra migliore comprensione del legame tra disturbi del metabolismo e aumento del rischio di cancro alla prostata. Dato che BM rigidità indotta dalla sua esposizione alle età può essere un innesco per l&#39;invasività in altro cancro tipi, sarà di interesse per utilizzare il protocollo per impostare modelli simili che incorporano normali cellule epiteliali e le cellule tumorali di altri organi (ad esempio seno, del colon, ovaie, pancreas). Passaggi critici all&#39;interno del protocollo, insieme con i loro tempi, sono riassunte nella Figura 4. Durante la fase iniziale, è fondamentale per mantenere la soluzione stock di RBM a 4 ° C, mentre si scioglie per evitare che la sua polymerization. puntali delle pipette non devono essere inseriti nella soluzione rM magazzino fino a quando non sono state refrigerate a 4 ° C. Per il passo successivo è anche importante per garantire i vetrini camera sono equilibrati a 4 ° C prima di essere rivestiti con la soluzione RBM. Appena la temperatura della soluzione MLF è aumentata oltre 4 ° C che a polimerizzazione irreversibile per formare un gel. È essenziale che i meccanismi non è disturbato durante la fase di polimerizzazione per assicurare che forma una superficie piana adatto per coltura cellulare e analisi microscopica. La durata di incubazione con GLA, con o senza inibitori della reazione di Maillard (sodiocianoboroidruro e amingoguanidine) determinerà come il gel rigido RBM diventa. Si consiglia di utilizzare una incubazione 6 ore con GLA se sono richieste condizioni di semi-rigide, e l&#39;incubazione 14 ore se sono necessarie condizioni rigide (Tabella 1). tempi di incubazione alternative o concentrazioni di GLA possono essere usati se diversi livellidi rigidità sono desiderato. In questo caso l&#39;analisi reologiche del gel RBM devono essere incorporato come un passo essenziale. Dopo la fase di tempra reazione di Maillard mediante incubazione con GEE e le successive fasi di lavaggio con PBS, i gel RBM possono essere utilizzati immediatamente o conservati a 4 ° C per un massimo di 48 ore prima del loro utilizzo per colture cellulari. Una volta colture cellulari sono impostati è importante cambiare il terreno di coltura (compresi eventuali trattamenti) ogni due giorni. Si raccomanda di mantenere le colture cellulari 3D per 3-12 giorni secondo i parametri oggetto di indagine. Per 3D PEC acini si consiglia di analizzare le culture dopo 6 giorni, e per il 3D PTC sferoidi analisi è raccomandata dopo 3 giorni di coltura in prima istanza. <img alt="Figura 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> Figura 4:. Semplice Panoramica del protocollo con i punti critici e tempi indicati la floSchema w descrive come preparare e irrigidire la membrana basale ricostituita (RBM) con glicolaldeide (reazione di Maillard) con i punti critici e tempi indicati. Punti in cui il protocollo può essere fermato, e gel RBM conservato, sono indicati anche. RBM, membrana basale ricostituita; GLA, glicolaldeide; GEE, glicina estere etilico; ON, durante la notte; PBS, fosfato tamponata salina; RT, a temperatura ambiente. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:628px;">I - Material for monolayer culture</td>
			<td style="width:265px;"></td>
			<td style="width:139px;"></td>
			<td style="width:401px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BPH-1</td>
			<td>CaP Cell Line Database</td>
			<td>PCaCL-132</td>
			<td>Contact: simon.hayward@mcmail.vanderbilt.edu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Complete keratinocyte serum-free media&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>17005-075</td>
			<td>Do not warm at 37 &#186;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum&#160;</td>
			<td>First Link UK Ltd</td>
			<td>02-00-850</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC3&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (Dulbecco A) tablets</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R5886</td>
			<td>warm in 37 &#186;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RWPE-1&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">II - Material for 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoguanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>396494</td>
			<td>Irritating to eyes, respiratory system and skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber slides, 8-well&#160;</td>
			<td>Thermo Scientific Nunc Lab-Tek</td>
			<td>TKT-210-816M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&#160;</td>
			<td>AMS Biotechnology</td>
			<td>3445-005-01</td>
			<td>Store Basement membrane (BM) at -80 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanogen bromide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C91492</td>
			<td>Toxic by contact skin and inhalation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine ethyl ester hydrochloride (GEE)</td>
			<td>Sigma-Aldrich</td>
			<td>50060</td>
			<td>Irritating to eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycolaldehyde dimer (GLA)</td>
			<td>Sigma-Aldrich</td>
			<td>G6805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium cyanoborohydride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71435</td>
			<td>Highly flammable; Toxic by contact skin and inhalation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filter 0.22 microns</td>
			<td>Appleton Woods</td>
			<td>BC680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">III - Material to quantify Maillard reaction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>ThemoFisher Scientific</td>
			<td>D3571</td>
			<td>light sensitive and store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cloning cylinders</td>
			<td>Sigma-Aldrich</td>
			<td>C1059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>ThemoFisher Scientific</td>
			<td>A-11001</td>
			<td>light sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>ThemoFisher Scientific</td>
			<td>A-11034</td>
			<td>light sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>Abcam</td>
			<td>ab7481</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting media&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-pentosidine clone PEN-12 mAb</td>
			<td>TransGenic Inc</td>
			<td>KH012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-human collagen IV polyclonal antibody&#160;&#160;</td>
			<td>Acris Antibodies</td>
			<td>R1041</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-laminin A/C pAb</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-7292</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol sorbitan monolaurate (Tween-20)</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis cassette Slide-A-Lyzer</td>
			<td>ThemoFisher Scientific</td>
			<td>66333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IV - Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ARG2 controlled strain rotational rheometer</td>
			<td>T.A. Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert S100 (20x magnification) microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td>
			<td>Solent Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Axiovert 200M (40x, 63x magnification) microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus LH50A microscope fitted with a digital camera using phase-contrast&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PHERAstar Plus plate reader spectrophotometer</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">V - Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image J 1.47v</td>
			<td>National Institute of Health, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaXpress</td>
			<td>Molecular Divices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Qui si descrive un protocollo che può essere utilizzato per studiare il microambiente biofisico relativi a spessore maggiorato e la rigidità della membrana basale (BM) durante le patologie legate all&#39;età e disturbi metabolici (come cancro, diabete, malattie microvascolare, retinopatia, nefropatia e neuropatia) . La premessa del modello è reticolazione non enzimatica della matrice ricostituito BM (RBM) di trattamento con glicolaldeide (GLA) promuovere avanzata glicazione prodotto finale (AGE) generazione tramite la reazione di Maillard. Esempi di tecniche di laboratorio che possono essere utilizzati per confermare la generazione AGE, reticolazione non enzimatica e una maggiore rigidità in GLA trattati meccanismi sono delineati. Questi includono la preparazione di natale RBM (trattato con soluzione salina tamponata con fosfato, PBS) e rigido RBM (trattato con GLA) per la determinazione di: suo contenuto AGE da analisi fotometrica e microscopia a immunofluorescenza, la sua reticolazione non enzimatica da sodio dodecil solfato poliacrilammideelettroforesi su gel (SDS PAGE) e microscopia confocale, e la sua maggiore rigidità utilizzando reometria. Il procedimento qui descritto può essere utilizzato per aumentare la rigidità (moduli elastici, E) di MLF fino a 3,2 volte, in linea con le misurazioni effettuate in sani contro tessuto prostatico umano malato. Per ricreare il microambiente biofisico associato con l&#39;invecchiamento e la prostata malata ghiandola tre tipi di cellule della prostata sono stati introdotti a RBM nativo e rigido RBM: RWPE-1, le cellule epiteliali della prostata (PEC) derivato da una ghiandola prostatica normale; BPH-1, PEC derivati ​​da una ghiandola prostatica affetti da iperplasia prostatica benigna (BPH); e PC3, cellule metastatiche derivate da un tumore osseo secondario proveniente da cancro alla prostata. parametri multipli possono essere misurati, tra cui le dimensioni, la forma e le caratteristiche invasive della acini ghiandolari 3D formato da RWPE-1 e BPH-1 sul nativo contro rigida RBM, e la durata media delle cellule, la velocità migratoria e la persistenza di movimento delle cellule di spher 3DOID formate da cellule PC3 alle stesse condizioni. Cellulare vie di segnalazione e la localizzazione subcellulare delle proteine ​​può anche essere valutato.

3D prostata Acini coltivate su Stiff RBM Dopo 6 giorni di coltura, PEC derivate da tessuto prostatico normale (RWPE-1) (Figura 1A) e il tessuto di BPH (BPH-1) (Figura 1B) forma acini su nativa (PBS trattata) RBM che sono organizzati in sferoidi uniformi epiteliale cellule. Questi acini hanno anche le caratteristiche di PEC altamente organizzati con spazio apicale-to-basale polarità e un luminale visibile 13,20. Il acini formata da PEC derivate da tessuto prostatico normale (RWPE-1) (Figura 1A) e tessuto di BPH (BPH-1) (Figura 1B) su irrigidito RBM (ricco-AGE) (trattato con GLA) hanno un&#39;architettura perturbato (shifting da sferoidale a forma poligonale e le cellule sporgenti / migrazione da acini nel RBM ricco di età) (Figura 1A). Queste<html> acini sono caratterizzati anche da PEC altamente disorganizzati che hanno perso la loro polarità apicale-to-basale con un piccolo o inesistente spazio luminale 13. <img alt="Figura 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> Figura 1:. Le cellule epiteliali della prostata coltivata come 3D ghiandolare Acini su Native e Stiff ricostituito membrana basale (RBM) (A) le immagini in campo chiaro di RWPE-1 le cellule coltivate per 12 ore fino a 6 giorni su gel meccanismi trattati con PBS (nativo) o 50 mm glicolaldeide per 14 ore (ricchi di età; rigida); Barra di scala = 50 micron. (B) BPH-1 le cellule, cresciute come descritto nel pannello A; Barre di scala = 50 micron; Dati rappresentativa 3 esperimenti indipendenti. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <html> Tabella 1 &quot;src =&quot; / files / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; Tabella 1:. Caratteristiche epiteliali della prostata RWPE-1 acini Grown su Native, semi-rigida e Stiff ricostituito membrana basale (RBM) RWPE-1 acini sono state coltivate su meccanismi pre-trattati con PBS per 14 ore (nativo), glicolaldeide (GLA ) per 6 ore (semi-rigido) o GLA per 14 ore (rigida). Per la forma acinar, la percentuale (%) ± deviazione standard (SD) di turno, acini semi-poligonale e poligonali sono stati calcolati da 5 esperimenti indipendenti (50 acini quantificati per condizione). dimensione acinare relativa è stata calcolata (nativo RBM = 100%) da 3 esperimenti indipendenti. Per invasività,% ± SD acini con una o più celle sporgenti sono stati calcolati da 3 esperimenti indipendenti. cambiamento Fold è calcolato dividendo il valore medio ottenuto in condizioni di semi-rigidi o rigide per il valore corrispondente per condizioni native. valori di P calcolati utilizzando il test t di Student (α = 0,05). ontent &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; AGE dipende maggiore rigidità RBM promuove la migrazione delle cellule tumorali della prostata PC3 Cellule PC3 coltivate su nativo RBM migrano, mantenendo costante il contatto cellula-cellula, mentre le cellule PC3 coltivate su ricchi di AGE (rigida) RBM mossa in modo indipendente l&#39;una dall&#39;altra (Figura 2A). Dopo 72 ore nelle cellule PC3 cultura formare foci (sferoidi) su nativo (PBS trattata) RBM, mentre le cellule PC3 sulla rigida (ricco di AGE) RBM Non da sferoidi e migrare in modo indipendente (Figura 2B). Cellule PC3 sulla rigida RBM (ricchi di AGE) sono più allungate rispetto alle cellule PC3 coltivate su nativo RBM (Figura 2C). Cellule PC3 sulla rigida meccanismi migrano più velocemente rispetto alle cellule PC3 coltivate su nativo RBM (Figura 2D). Cellule PC3 sulla rigida meccanismi mostrano una diminuzione della persistenza rispetto alle cellule PC3 coltivate su nativo RBM (Figura 2E). <img alt = &quot;Figura 2&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig2.jpg&quot; /&gt; Figura 2:. La migrazione delle cellule tumorali della prostata su Native e Stiff ricostituito membrana basale (RBM) (A) le immagini in campo chiaro di cellule PC3 coltivate su gel di meccanismi trattati con PBS (nativo) o 50 mm glicolaldeide per 14 ore (ricchi di AGE, rigido) . Le cellule sono state ripreso con un microscopio campo chiaro (obiettivo 10X) e un tasso di acquisizione di 1 immagine per ora per 12 ore seguita da cellule di monitoraggio per generare traiettorie. Immagini mostrati corrispondono ai punti di tempo dopo 0, 3, 6, 9 e 12 ore. Traiettorie delle singole celle sono indicati per il punto di tempo 12 hr. Barra di scala = 100 micron. Cellule (B) PC3 coltivati ​​su meccanismi nativo o rigida per 72 ore, e ripreso come descritto nel pannello (A). Barra di scala = 100 micron. Particolare mostra area selezionata a 2X ingrandimento. (C) Media ± SD lunghezza cellulare (micron); differenza significativa tra i meccanismi nativi e rigido RBM (p = 1.2 x 10 -23). ( <strong&gt; D) Media ± SD velocità (micron / ora) calcolato da traiettorie delle cellule; differenza significativa tra i meccanismi nativi e rigido RBM (p = 0,004). (E) Media ± SD persistenza del movimento delle cellule (rapporto D / T, dove D = distanza dall&#39;inizio alla fine della traiettoria delle cellule, T = lunghezza totale della traiettoria delle cellule); differenza significativa tra i meccanismi nativi e rigido RBM (p = 0,0007). Per i pannelli CE&gt; 10 cellule sono stati analizzati, i dati rappresentativa 3 esperimenti indipendenti. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Qui spieghiamo un protocollo per modellare il microambiente biofisico dove reticolazione e una maggiore rigidità della membrana basale (BM) indotta da prodotti finali della glicazione avanzata (AGE) ha rilevanza patologica.

Come studiare membrana basale Rigidità come biofisica trigger nel cancro alla prostata e altre patologie legate all&#39;età o malattie metaboliche

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

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Cancer Research

9.6K Views.  Universidad de la República (UDELAR).  Qui spieghiamo un protocollo per modellare il microambiente biofisico dove reticolazione e una maggiore rigidità della membrana basale (BM) indotta da prodotti finali della glicazione avanzata (AGE) ha rilevanza patologica. 

Video: How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Coculture saggi per studiare macrofagi e microglia stimolazione del glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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Ricerca sul cancro

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Il<em&gt; Drosophila</em&gt; Immaginale Disc tumore Modello: visualizzazione e la quantificazione di espressione genica e di Tumor invasività Utilizzando genetica Mosaici

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

hyperpolarized<sup&gt; 13</sup&gt; C metabolica spettroscopia di risonanza magnetica e Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Il Colon-26 Carcinoma tumore-cuscinetto mouse come un modello per lo studio del cancro cachessia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Rilevamento del potenziale di membrana di mitocondri per studiare CLIC4 HN4 Knockdown-indotta delle cellule apoptosi In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Fosfopeptide arricchimento accoppiata alla spettrometria di massa privo di etichetta quantitativa per indagare il Phosphoproteome nel carcinoma della prostata

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Il lievito come telaio per lo sviluppo di saggi funzionali per studiare l'Uomo P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Endpoint importanti e marcatori proliferativi per valutare piccole lesioni intestinali e adattamento utilizzando un modello murino di mucosite indotta dalla chemioterapia

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In Vitro Analisi per studiare l'interazione tumora-macrophage

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Come studiare membrana basale Rigidità come biofisica trigger nel cancro alla prostata e altre patologie legate all'età o malattie metaboliche

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