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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The authors have nothing to disclose.

Saf RBM jeller 6 MECS 3D ​​glandüler asinüs üretilmesi için bir protokol I RBM matrise kolajen 4 mg / ml tip eklenmesiyle yapılan bir çalışmada değiştirildi. kollajen eklenmesi 1589 ± 380 Pascal 175 ± 37 artan RBM jel elastik modül ile sonuçlanmıştır. Sertlik Bu 9.1 katlık bir artış MECS 21 büyüme, hayatta kalma, göç ve farklılaşmayı modüle. - (-) - RBM jele ilave edilmiştir tip I kollajenin enzimatik olmayan çapraz bağlanmasını teşvik etmek riboz Protokol D ile muamele aşaması içerecek şekilde tekrar modifiye edildi. Sertlik Elde edilen 15 kat artış onların invaziv davranış 22 teşvik etmek MECS onkojenik dönüşümü ile işbirliği bulunmuştur. Ben RBM jeller kollajenin ekleme Çeşidi deneysel yaklaşım sadece stroma arasındaki fiziksel bariyer sonra insan dokusunda meydana kolajen lifleri ile MECS doğrudan etkileşimi kolaylaştırırve BM sağladığı epitel proteolitik bozulmaya uğrar. Saf RBM jeller GLA ile ön işlemden de Halk 3D bez asinüsü üreterek, mevcut protokol BM sertliği başına onların invaziv davranış (Şekil 3) tetikleyebilir nasıl incelemek için bir yol açar. Bu protokol kaynaklı BM sertlik seviyeleri fizyolojik alaka var. 6 saat, 14 saat süre ile, 50 mM GLA ile inkübasyon, sırasıyla PBS (Tablo 1) ile muamele RBM jeller 122 ± 55 Pascal göre 175 ± 90 ve 322 ± 160 saf RBM jel elastik modülü artmıştır. RBM sertliği bu 1,7-3,2 misli artış normal prostatta ve BPH dokusunun 23-26 ile karşılaştırıldığında habis gözlenen sertlik 2.5- 3.4-kat artış tartışıldı. Son yayında PEC asini YAŞ ve RBM sertlik birikimi ile oluşturulan 13 morfolojik değişiklikler belirtildiği gibi ar dan istatistiksel olarak anlamlı vardiyası için sayısal olabilirçokgen şekline ounded luminal / toplam asinar alan ve yaş bakımından zengin RBM (Şekil 3) içerisine acina taşıma çıkıntı yapan hücrelerin azalmıştır. İmmünoblotting da EMT belirteçleri değerlendirmek ve kasılma davranışı (örneğin fosforlu miyosin hafif zincir-2, pMLC2 13) sert RBM normale karşı yetiştirilen Halk (Şekil 3) (E-kadherin 13 örneğin kaybı) kullanılabilir. 130: ve GM130 erken endozomal antijeni 1: immünofloresan boyama ve konfokal mikroskopi kullanılarak daha fazla değerlendirilmesi (örneğin, laminin, kolajen IV ve AGE birikimi 13), hücresel apikal-to-bazal polarite (örneğin, apikal EEA1 lokalizasyonu BM görselleştirmek için uygulanabilir kDa sis-Golgi işaretleyici 13) ve yapışma moleküllerinin hücre modelleri (hücre-hücre kavşaklarından 13 örneğin e-kaderin yerelleştirme) (Şekil 3). <img alt = &quot;Şekil 3&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig3.jpg&quot; /&gt; Şekil 3:. Burada sunulan Farklı Protokoller genel şeması (hazırlamak ve glikoladehid (Maillard reaksiyonu) ile yeniden bazal membran (RBM), sert RBM için hücreleri tohum nasıl nasıl sert RBM analiz pekiştirmek için nasıl tasvir Maillard reaksiyonu), ve yaş bakımından zengin RBM neden olduğu hücresel ve moleküler değişimleri analiz etmek için kullanılabilir prosedürlerin ölçüde. YAŞ, gelişmiş Glikasyon Son; BM, bazal membran; DAPI, 4 &#39;, 6-diamidino-2-fenilindol; EEA1 erken endozomal antijeni 1; GAPDH, gliseraldehid-3-fosfat dehidrojenaz, GLA izleyerek, sulu; GEE glisin etil ester; GM130, 130 kDa&#39;lık bir sis-Golgi işaretleyici; p-MLC2 (Thr18 / Ser19), miyozin hafif zincir-2 siteleri fosforile 18 ve serin 19 treonine; RBM, tekrar oluşturulmuş temel membran; SDS-PAGE sodyum dodesil sülfat poliakrilamid jel elektroforezi. RWPE1 acininin Ölçek çubuğu = 10 mm; PC3 tümör hücresi içinSferoidler Ölçek çubuğu = 100 mikron. Bu rakam referans 13 modifiye edilmiştir. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> - (-) - Riboz RBM için çapraz bağlama maddesi olarak seçilmiştir D ise giderme adımları gereklidir. - (-) - 72 saat boyunca riboz, daha önce RBM / kollajen jel 22 için tarif edildiği gibi, RBM jeller dehidrasyon ve çekme ile sonuçlanmıştır protokolü gelişimi sırasında, 1 M, D ile tedavi olduğu bulunmuştur. - (-) - D düşük konsantrasyonlarda değerlendirilmesi riboz ve daha kısa tedavi süreleri bu sınırlamayı aşmak için yardımcı olabilir. RBM sertlik yüksek seviyelerde arzu nerede protokol gelecekteki uygulamalar bir potansiyel sınırlama karşılaştı olabilir. uzun inkübasyon süreleri ve GLA&#39;nın daha yüksek konsantrasyonlar RBM jel sertliği yüksek seviyelerini indüklemek için, kullanılacak ise gerekli olacaktır<html> Bu tedavi koşulları hücrenin hayatta kalması ve çoğalması üzerinde bir etkisi olup olmadığını önceden 13 açıklandığı gibi, değerlendirmek. Ayrıca, serum ile RWPE-1 hücrelerinin inkübasyon kaçınılmalıdır serum veya serum ihtiva eden malzeme, bir fenotipik EMT benzeri geçiş ve maruz indükler unutulmamalıdır. Deneyler kısa RNA (siRNA) oligonükleotidleri müdahale transfeksiyonunu içeren, örneğin, işlemin düşük serum transfeksiyon ortamı hücrelerin geçmeden KSFM yetiştirilen RWPE-1 hücreleri kullanılarak optimize edilmelidir. geçici siRNA kullanılarak modelde yaklaştığında genin seviyesini tehlikeye atabilecek Bu dezavantajı elde susturulması. Bazı protein hedefleri için ayarlanabilir gen susturma ve protein düzeyleri istenen düşüşün indüklenebilir shRNA vektörler istihdam için tavsiye edilir. Stromal hücre veya tümör hücresi bağlantılı lisil oksidaz (LOX) 17 enzimatik çapraz bağlanmasını dahil uyarlamalar da gelecek modelleri içine dahil edilebilir. jove_content &quot;&gt; Bu protokol yanlısı invaziv Halk AGE-bağımlı BM sertliği tarafından tetiklenen mekanizmaların (RWPE-1, BPH-1) ve invaziv PTCs (PC3) anti-metastatik hedeflerin değerlendirilmesi gelecekteki çalışma kolaylaştıracaktır. BPH göz önüne alındığında bir metabolik bozukluk 27 olarak kabul edilir, bu protokol aynı zamanda metabolik bozukluklar ve artan prostat kanseri riski arasındaki bağlantıyı bizim daha iyi anlaşılması yönünde önünü açıyor. AGEs onun maruz tarafından uyarılan BM sertlik diğer kanser invazivlik için bir tetikleyici olabilir göz önüne alındığında türleri, diğer organların (örneğin meme, kolon, yumurtalık, pankreas) normal epitel hücreleri ve tümör hücrelerini birleştirmek benzer modeller kurmak için protokolünü kullanmak için ilgi olacaktır. Birlikte zamanlamaları ile bir protokol dahilinde kritik adımlar, Şekil 4&#39;te özetlenmiştir. İlk aşamasında o onun pol önlemek için thaws ise 4 ° C&#39;de RBM stok solüsyonu korumak için esastırymerization. bunlar, 4 ° C&#39;ye soğutulmuş kadar pipet uçları rM hazır çözelti içine yerleştirilmesi gerekir. Bir sonraki adım için bunların RBM çözeltisi ile kaplanır önce bölmeli lamlar 4 ° C&#39;ye dengelenmiş emin olmak için önemlidir. En kısa RBM çözeltinin sıcaklığı, 4 ° C üzerinde bir artış gibi bir jel oluşturmak için geri dönüşü olmayan bir polimerizasyon geçirecektir. Temel RBM bir daha da hücre kültürü ve mikroskobik analizi için uygun bir yüzey oluşturur sağlamak için polimerleştirme aşamasında rahatsız olduğunu. veya Maillard reaksiyonu (sodyum siyanoborohidrit ve amingoguanidine) önleyicileri olmayan GLA ile inkübasyon süresi RBM jel haline kadar sert belirleyecektir. Sert koşullar (Tablo 1) gerekiyorsa Yarı sert koşullar gerekiyorsa GLA ile 6 saat inkübasyon kullanılması tavsiye ve 14 saat inkübasyon edilir. GLA Alternatif inkübasyon süreleri veya konsantrasyonları farklı düzeylerde ise kullanılabilirsertliğin arzu edilir. RBM jellerinin Bu durumda, reolojik analizde temel bir adım olarak dahil edilmesi gerekir. GEE ile inkübasyon ve PBS ile müteakip yıkama adımları ile Maillard reaksiyonu söndürülmesi adımını takiben, RBM jeller hemen kullanılabilir ya da önceki hücre kültürü için kullanımı ile en fazla 48 saat boyunca 4 ° C&#39;de saklanır. hücre kültürleri ayarlandıktan sonra her iki gün (herhangi bir tedavi de dahil olmak üzere), kültür ortamı değiştirmek önemlidir. Araştırma altındaki parametrelerine göre 3-12 gün için 3 boyutlu hücre kültürleri korumak için tavsiye edilir. 3D için PEC 6 gün sonra kültürleri analiz etmek tavsiye ve 3D PTC ilk etapta kültür 3 gün sonra tavsiye edilir analiz sferoidler için olan asinüsü. <img alt="Şekil 4," src="/files/ftp_upload/54230/54230fig4.jpg" /> Şekil 4:. Kritik Adımlar ve Zamanlamaları&#39;na ile Protokol Basit Bakış gösterilen flow şeması hazırlamak ve belirtilen kritik adımlar ve zamanlamaları ile glikoladehid (Maillard reaksiyonu) ile yeniden bazal membran (RBM) pekiştirmek için nasıl göstermektedir. Protokol durdurulabilir ve RBM jeller saklanan Nokta de belirtilmiştir. RBM, tekrar oluşturulmuş temel membran; GLA izleyerek, sulu; GEE glisin etil ester; AÇIK, gecede; PBS, fosfat tamponlu salin; RT, oda sıcaklığı. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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

Burada, yaşa bağlı patolojiler ve metabolik hastalıklar (örneğin, kanser, diyabet, mikrovasküler hastalık, retinopati, nefropati ve nöropati) sırasında artan kalınlığı ve bazal membran (BM) sertliği ile ilgili biyofiziksel mikro-incelemek için kullanılabilecek bir protokol açıklar . modelin öncül Maillard reaksiyonu ile ileri glikasyon son ürün (AGE) üretimini teşvik etmek için glikoladehid (GLA) ile işlenmesi suretiyle yeniden BM (RBM) matrisin enzimatik olmayan çapraz bağlar. YAŞ nesil, non-enzimatik çapraz bağlanmasını onaylamak için kullanılır ve GLA sertliği artırılabilir laboratuvar teknikleri örnekleri RBM özetlenmiştir tedavi. fotometrik analiz ve immünoflüoresan mikroskopi, sodyum dodesil sülfat poliakrilamid tarafından enzimatik olmayan çapraz bağlama ile onun yaş içerik: Bu belirlenmesi için (GLA ile muamele edilmiş) ve sert RBM (fosfat tamponlu tuzlu su, PBS ile muamele edilmiş) yerel RBM hazırlanmasını içerirjel elektroforezi (SDS-PAGE) ve konfokal mikroskopi, ve artan sertlik ile reometre. Burada tarif edilen prosedür, hastalıklı insan prostat dokusu karşı sağlıklı yapılan ölçümler ile uyumlu olarak, 3.2 kata kadar RBM sertliğini (esneklik modülü, E) geliştirmek için de kullanılabilir. yaşlanma ve hastalıklı prostat ile ilişkili biyofiziksel mikro yeniden üç prostat hücre tipleri yerli RBM ve sert RBM için getirilmiştir bezi: RWPE-1, prostat epitel hücreleri (PEC) normal prostat bezinin türetilmiş; BPH-1, benign prostat hiperplazisi (BPH) etkilenen bir prostat bezinin türetilen PECS; ve PC3, metastatik prostat kanseri hücreleri kaynaklanan ikincil kemik tümörü türetilmiş. Birden çok parametre boyut, şekil ve sert RBM karşı doğal ilgili RWPE-1 ve BPH-1 tarafından oluşturulan 3D glandüler asinüs invaziv özellikleri ve ortalama hücre boyu, göç eden hız ve 3D spher hücre hareketinin devam etmesi de dahil olmak üzere, ölçülebiliraynı koşullar altında, PC3 hücrelerinin oluşturduğu Giller. yollar ve proteinlerin hücre içi lokalizasyonu hücre sinyal de değerlendirilebilir.

Sert RBM 3D Prostat asinüsü Kültür Kültürde 6 gün sonra, Halk, normal prostat dokusu elde edilen (RWPE-1) (Şekil 1 A) Prostat dokusu (SPH-1) epitel düzgün sferoidler şeklinde düzenlenir (Şekil 1B) yerli (PBS ile tedavi edilmiş) RBM form acininin hücreler. Bu acininin da kutuplarına-bazal-apikal ve görünür bir lümen alanı 13,20 ile son derece organize Halk özelliklerine sahip. Normal prostat dokusu elde Halk oluşturduğu acininin (RWPE-1) (Şekil 1 A) ve (GLA ile muamele edilmiş) sertleştirilmiş (AGE zengin) RBM BPH dokusunun (BPH-1), (Şekil 1B), bir bozulmuş bir mimariye sahip (vites şekil ve hücrelerde çokgen için sfero itibaren) AGE zengin RBM) (Şekil 1A içine asinüs göç / çıkıntılı. Bunlar<html> acininin aynı zamanda küçük veya hiç lümen boşluk 13 ile apikal-to-bazal polarite kaybetmiş yüksek dağınık Halk ile karakterize edilir. <img alt="Şekil 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> Şekil 1:. Ana ve sert tekrar oluşturulmuş temel membran (RBM) 3D Bez asinüs olarak yetiştirilir Prostat Epitelyal hücreler (A), RWPE-1 hücrelerinin aydınlık ve görüntüler, PBS (ana) ya da işlemden RBM jelleri üzerinde 6 gün, 12 saat kadar yetiştirilen 14 saat (; sert AGE-zengin) için 50 mM glikoladehid; Ölçek çubuğu 50 mikron =. Panel A&#39;da açıklandığı gibi büyütüldü (B) BPH-1 hücreleri; Ölçek çubukları 50 mikron =; Veri 3 bağımsız deneyler temsilcisidir. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <html> Tablo 1 &quot;src =&quot; / files / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; Tablo 1:. Yerli Yarı Sert ve sert tekrar oluşturulmuş temel membran (RBM) Yetiştirilen Prostat Epitelyal RWPE-1 asinüs özellikleri RWPE-1 acininin GLA (RBM 14 saat (yerli) izleyerek, sulu PBS ile önceden tedavi edilen büyütüldü ) 6 saat (yarı-sert) veya 14 saat (sert) için GLA. asiner şekli için, yüzde (%) yuvarlak standart sapma (SD) ±, yarı köşeli ve çokgen acininin (50 acininin durum başına miktarı) 5 bağımsız deneyden hesaplandı. Bağıl asiner boyutu 3 bağımsız deneyler (yerli RBM =% 100) hesaplandı. invaziflik için, bir veya daha fazla çıkıntı yapan hücrelerin SD asinüs ±% 3 bağımsız deney ile hesaplandı. Katlama değişim doğal koşullar için karşılık gelen değeri ile yarı sert veya sert koşullar altında elde edilen ortalama değer bölünmesi ile hesaplanır. P değerleri, Öğrenci t-testi (α = 0.05) kullanılarak hesaplandı. ontent &quot;fo: keep-together.within sayfa =&quot; 1 &quot;&gt; YAŞ bağımlı artmış RBM sertlik PC3 prostat tümörü hücre göçü teşvik PC3 hücreleri AGE-zengin (sertleßmiß) üzerinde büyümüş, oysa yerel RBM yetiştirilen PC3 hücreleri birbirlerine (Şekil 2A) için bağımsız olarak, sürekli hücre-hücre teması sağlanarak RBM hareket geçirmek. Kültür PC3 hücreleri 72 saat yerli üzerine odaklar (sferoidler) oluşturmak sonra sferoidler değil yapmak ve bağımsız (Şekil 2B) göç sert (YAŞ-zengin) RBM üzerinde PC3 hücreleri ise, RBM (PBS tedavi). Sert (YAŞ-zengin) RBM üzerinde PC3 hücreleri yerli RBM (Şekil 2C) yetiştirilen PC3 hücreleri daha uzundur. Sert RBM üzerinde PC3 hücreleri yerli RBM (Şekil 2B) yetiştirilen PC3 hücreleri daha hızlı göç ederler. Sert RBM ilgili PC3 hücreleri doğal RBM (Şekil 2E) üzerinde büyümüş PC3 hücreleri ile karşılaştırıldığında kalıcılık olarak bir azalma sergilerler. <img alt = &quot;Şekil 2&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig2.jpg&quot; /&gt; Şekil 2:. Yerli ve Sert Sulandırılmış Bodrum Membran (RBM) üzerine Prostat Tümör Hücre Göç (A) 14 saat süreyle PBS (yerli) veya 50 mM glikoladehid ile tedavi RBM jeller üzerinde yetiştirilen PC3 hücreleri aydınlık görüntüleri (YAŞ zengin, sert) . Hücreler, bir aydınlık mikroskop (10x objektif) ve yörüngeleri üretmek için takip hücre ardından 12 saat boyunca saat başına 1 resim bir alıcı oranı kullanılarak görüntülendi. Gösterilen görüntüler, 0, 3, 6, 9 ve 12 saat sonraki zaman noktalarında karşılık gelmektedir. tek hücre yörüngeleri 12 saat bir zaman noktası için gösterilmiştir. Ölçek çubuğu = 100 mikron. Panel (A) &#39;da tarif edildiği gibi (B)&#39; PC3 hücreleri 72 saat için yerli veya sert RBM kültüre ve görüntüsü alınmıştır. Ölçek çubuğu = 100 mikron. Detay 2X büyütme seçilen alanı göstermektedir. (C) Ortalama ± SD hücre uzunluğu (um); yerli RBM ve sert RBM arasında anlamlı bir fark (p = 1.2 x 10 -23). ( <strHücre yörüngeleri hesaplanan ong&gt; D) Ortalama ± SD hızı (mm / saat); yerli RBM ve sert RBM (p = 0.004) arasında anlamlı fark. Hücre hareketi (E) Ortalama ± SD sebat (oran D / T, burada D = baştan hücre yörünge, hücre yörünge T = toplam uzunluğu sonuna mesafe); yerli RBM ve sert RBM (p = 0.0007) arasında anlamlı fark. CE&gt; 10 hücre analiz edildi paneller için, veri 3 bağımsız deneyler temsilcisidir. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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

Burada çapraz ve ileri glikasyon nihai ürünlerin (AGE) tarafından uyarılan bazal membran (BM) artan sertlik patolojik ilgisi biyofiziksel mikro modelleme için bir protokol açıklar.

Prostat Kanseri ve Diğer Yaşa bağlı Patolojilerde veya Metabolik Hastalıklarda bir Biyofiziksel Tetik olarak Bazal Membran Rijitliğinin Eğitim Nasıl

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

how-to-study-basement-membrane-stiffness-as-biophysical-trigger

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JoVE Journal

Cancer Research

9.6K Görüntüleme.  Universidad de la República (UDELAR).  Burada çapraz ve ileri glikasyon nihai ürünlerin (AGE) tarafından uyarılan bazal membran (BM) artan sertlik patolojik ilgisi biyofiziksel mikro modelleme için bir protokol açıklar. 

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 Tahliller Glioblastoma Invasion Makrofaj ve Mikroglia Stimülasyon Eğitim için

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

Kanser Araştırmaları

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.

<em&gt; Drosophila</em&gt; Hayali Disk Tümör Modeli: Genetik Mozaikler kullanma Gen İfade ve Tümör invazivliğinin Görselleştirme ve

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 Metabolik Manyetik Rezonans Spektroskopisi ve Görüntüleme

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.

Kolon-26 Karsinoma Tümör taşıyan Kanser Kaşeksi Araştırmaları Modeli Olarak Fare

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.

Mitokondri zar potansiyeli CLIC4 nakavt kaynaklı HN4 hücre Apoptosis Vitro çalışma tespiti

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.

Prostat kanserinde Phosphoproteome araştırmak için etiket içermeyen nicel kütle spektrometresi ile birleştiğinde Phosphopeptide zenginleştirme

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.

Insan p53 çalışma fonksiyonel Assays geliştirmek için bir şasi olarak Maya

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.

Kemoterapiye bağlı Mukozinin fare modelini kullanarak küçük bağırsak yaralanmasını ve uyarlamasını değerlendirmek için önemli uç noktalar ve proliferatif Işaretçiler

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 assay tümör çalışma-macrophage etkileşim

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.

Prostat Kanseri ve Diğer Yaşa bağlı Patolojilerde veya Metabolik Hastalıklarda bir Biyofiziksel Tetik olarak Bazal Membran Rijitliğinin Eğitim Nasıl

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