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.

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The authors have nothing to disclose.

순수 RBM 젤 6 MEC의에서 3D 선의 선포 세포의 생성을위한 프로토콜은 내가 RBM 행렬에 콜라겐 4 ㎎ / ㎖ 유형의 추가에 의해 이전의 연구에서 수정되었습니다. 콜라겐의 첨가는 1,589 ± 380 ± 175 파스칼 (37)로부터 증가 RBM 겔 탄성률 결과. 강성이 9.1 배 증가 MEC의 (21)의 성장, 생존, 마이그레이션 및 차별화를 변조. - (-) - RBM 겔에 첨가 한 타입 I 콜라겐의 비 효소 적 가교 결합을 촉진하기 리보스 프로토콜은 D와 처리 공정을 포함하여 다시 변경되었다. 강성의 결과로 15 배 증가 그들의 침략 행위 (22)을 촉진하기 위해 MEC의의 종양 변화와 협력 밝혀졌다. I가 RBM 겔 콜라겐 추가 유형의 실험 방법에만 기질 간의 물리적 배리어 후 인체 조직에서 발생하는 콜라겐 섬유와 MEC의 직접적인 상호 작용을 용이상기 BM에 의해 제공 상피 단백질 가수 분해를 거친다. 순수 RBM 젤 GLA로 사전 처리의 농도 (PEC)에서 3D 선의 선포 세포를 생성하여, 현재의 프로토콜은 BM 강성 자체가 자신의 침략 행위 (그림 3)을 실행할 수있는 방법을 연구하는 방법을 엽니 다. 이 프로토콜에 의한 BM 강성의 수준은 생리 학적 관련성을 가지고있다. 6 시간, 14 시간 동안 50 mM의 GLA와 배양은 각각 PBS (표 1)로 처리 RBM 젤 122 ± 55 파스칼에 비해 175 ± 90, 322 ± 160 순수 RBM 젤의 탄성 계수 증가했다. RBM의 강성이 1.7-3.2 배 증가는 정상 전립선 또는 BPH 조직 23-26에 비해 악성 관찰 강성에서 2.5로 3.4 배 증가 되풀이되었습니다. 최근 책에서 PEC 꽈리의 나이와 RBM 강성의 축적에 의해 유발 13 형태 학적 변화를 설명 된대로 아칸소에서 통계적으로 유의 한 변화에 대한 정량화 할 수있다다각형 모양에 ounded, 내강 / 총 선포 영역 및 AGE 풍부한 RBM (그림 3)에 acina에서 이주 돌출 세포를 감소. 면역 또한 EMT의 마커를 평가하기 위해 상기 수축 거동 (예 인산화 미오신 경쇄 -2 pMLC2 13) 강성 RBM 대 정상 성장 농도 (PEC)에서 (도 3) (E-cadherin의 (13)의 소멸)을 사용할 수있다. 130 : 및 GM130 초기 엔도 좀 항원 1 : 면역 염색 및 공 초점 현미경을 사용하여 추가 평가 (예를 들어, 라미닌, 콜라겐 IV와 AGE 축적 13), 휴대 혀끝 - 투 - 기초 극성 (예를 들어 정점 EEA1의 현지화 BM을 시각화에 적용 할 수 kDa의 시스 - 골지 마커 13)과 접착 분자의 세포 패턴 (세포 - 세포 접합 (13)에 예를 들어, E-cadherin의 현지화) (그림 3). <img aLT = &quot;그림 3&quot;SRC = &quot;/ 파일 / ftp_upload / 54230 / 54230fig3.jpg&quot;/&gt; 그림 3 :. 여기에 제시된 다른 프로토콜의 개요도 (준비 글리콜 알데히드 (메일 라드 반응)으로 재구성 기저막 (RBM), 뻣뻣한 RBM에게 세포를 시드하는 방법, 뻣뻣한 RBM을 분석을 단단하게하는 방법을 보여줍니다 메일 라드 반응) 및 AGE 풍부한 RBM에 의해 유도 된 세포 및 분자 변화를 분석 할 수있는 절차 정도. AGE, 당화 산물; BM, 기저막; DAPI, 4 &#39;, 6-diamidino -2- 페닐 인돌; EEA1, 초기 엔도 좀 항원 (1); GAPDH, 글리 세르 알데히드 -3- 포스페이트 데히드로게나; GLA, 글리콜 알데히드; GEE 글리신 에틸 에스테르; GM130, 130 kDa의 시스 - 골지 마커; P-MLC2 (Thr18 / Ser19), 미오신 경쇄-이 사이트에서 인산화 (18)과 세린 (19) 트레오닌; RBM, 재구성 기저막; SDS-PAGE 나트륨 도데 실 설페이트 폴리 아크릴 아미드 겔 전기 영동. RWPE1 꽈리 스케일 바 = 10 μm의; PC3 종양 전지용타원체 스케일 바 = 100 μm의. 이 그림은 참조 (13)에서 수정되었습니다. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> - (-) - 리보스 RBM위한 가교제로서 선택되는 D가 표시된 경우 단계가 필요할 것이다. (- -) - 72 시간 동안 리보오스, 이전에 RBM / 콜라겐 젤 (22)에 기재 한 바와 같이, RBM 젤의 탈수 및 수축의 결과 프로토콜 개발 과정은 1 M의 D와 치료를 한 것으로 나타났습니다. - (-) - D의 낮은 농도의 평가 리보스 짧은 치료 시간은 이러한 한계를 극복하는 데 도움이된다. RBM 강성의 높은 수준이 요구되는 경우, 프로토콜의 미래의 애플리케이션에 제한 전위가 발생 될 수있다. 긴 배양 시간 및 GLA의 높은 농도가 RBM 겔 강성의 높은 수준을 유도하기 위해 사용되는 경우가 필요할 것이다<html> 이러한 처리 조건은 세포 생존 및 증식에 영향을 줄 수 있는지 여부를 미리 13 바와 같이 평가한다. 또한 혈청 RWPE-1 세포의 배양은 피해야 혈청 또는 혈청 - 함유 물질에 대한 표현형 EMT 같은 전이 노광을 유도함을 유의해야한다. 실험 짧은 RNA (siRNA의) 뉴클레오티드 간섭의 형질 전환을 포함하는 경우, 예를 들어, 절차는 낮은 혈청 형질 감염 배지로 세포를 전환하지 않고, KSFM 성장 RWPE-1 세포를 이용하여 최적화되어야한다. 일과성의 siRNA를 사용하는 모델에 접근 할 때 유전자 수준을 손상시킬 수있는 단점이 달성의 음소거. 일부 단백질 표적을 위해 그것은 조정 가능한 유전자 침묵과 단백질 수준에서 원하는 감소를위한 유도의 shRNA 벡터를 사용하는 것이 좋습니다 것입니다. 간질 세포 또는 종양 세포 관련 리실 산화 효소 (LOX) (17)에 의해 효소 가교를 통합 적응은 미래 모델에 통합 될 수있다. jove_content은 &quot;&gt;이 프로토콜은 프로 침습적 농도 (PEC)에 AGE 의존 BM 강성에 의해 트리거 메커니즘 (RWPE-1, BPH-1) 및 침입 PTCS (PC3)에 반 전이 대상의 평가의 미래 연구를 촉진 할 것이다. BPH 점을 감안 대사 장애 (27)로 간주됩니다,이 프로토콜은 또한 신진 대사 장애 및 증가 된 전립선 암 위험 사이의 링크의 우리의 개선 된 이해를 향해 길을 불법 체류자.은 AGEs에의 노출에 의해 유도 된 BM 강성이 다른 암에 침입하기위한 트리거가 될 수 있음을 감안할 때 유형은, 다른 기관 (예를 들어 유방, 결장, 난소, 췌장)에서 정상 상피 세포와 종양 세포를 포함 유사한 모델을 설정 프로토콜을 사용하여 관심을 가질 것이다. 함께 그들의 타이밍과 프로토콜 내에서 중요한 단계는도 4에 요약되어있다. 초기 단계에서는 그것의 POL 방지 해동하면서 4 ° C에서 RBM의 원액을 유지하는 것이 필수적이다ymerization. 그들은 4 ° C로 냉각 될 때까지 피펫 팁은 RM은 원액에 배치 할 수 없습니다. 다음 단계는 그들이 RBM 용액 코팅 전에 상기 챔버 슬라이드는 4 ℃에 평형화 보장하기 위하여 중요하다. 즉시 RBM 용액의 온도는 39 ° C의 이상 증가로는 겔을 형성하기 위해 비가역 중합을 거칠 것이다. 이는 필수적인 RBM은 짝수 세포 배양 및 현미경 분석에 적합한 표면을 형성하도록 중합 단계 동안 방해되지 않도록. 또는 메일 라드 반응 (시아 노 수소화 붕소 나트륨과 amingoguanidine)의 억제제없이 GLA와 인큐베이션 기간은 RBM 겔 방법 뻣뻣하게 결정할 것이다. 뻣뻣한 조건 (표 1) 필요한 경우는 반 강성 조건이 필요한 경우 GLA와 함께 6 시간 배양를 사용하는 것이 좋습니다, 14 시간 배양한다. GLA의 다른 배양 시간 또는 상이한 농도 수준 경우 사용될 수있다강성 요구된다. RBM 겔이 경우 유동 학적 분석에서 필수적인 단계로 통합 될 필요가있다. GEE 함께 배양하고 PBS로 후속 세척 단계에 의해 메일 라드 반응을 급냉하는 단계에 이어, RBM 겔 바로 사용되거나 종래의 세포 배양을위한 그들의 용도에 최대 48 시간 동안 4 ℃에서 보관. 세포 배양이 설정된 후에는 이틀 (모든 치료 포함) 배지를 변경하는 것이 중요하다. 조사중인 파라미터에 따라 3~12일 대한 3 차원 세포 배양 물을 유지하도록 권장한다. 3D를 들어 PEC는 6 일 후에 문화를 분석하는 것이 좋습니다 및 3D PTC는 첫 번째 인스턴스에서 배양 3 일 후 권장 분석을 회전 타원체위한 것입니다 꽈리. <img alt="그림 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> 그림 4 :. 중요한 단계 및 타이밍과 프로토콜의 간단한 개요 표기됩니다 플로w도 준비하고 표시된 중요한 단계 및 타이밍과 글리콜 알데히드 (메일 라드 반응)으로 재구성 기저막 (RBM)를 단단하게하는 방법을 보여줍니다. 프로토콜이 중단 될 수 있고, RBM 겔 저장 지점도 표시된다. RBM, 재구성 기저막; GLA, 글리콜 알데히드; GEE 글리신 에틸 에스테르; ON, 밤새; PBS, 인산염 완충 염수; RT, 실내 온도가. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

여기서 우리는 연령과 관련된 병리 및 대사성 질환 (예를 들면 암, 당뇨병, 미세 혈관 질환, 망막증, 신장 병증 및 신경 병증) 중에 증가 된 두께 및 기저막 (BM)의 강도에 관련된 생물 물리학 적 미세 환경을 연구하는데 사용될 수있는 프로토콜을 설명 . 모델의 전제는 메일 라드 반응 (Maillard reaction)을 통해 당화 최종 생성물 인 (AGE) 생성을 촉진하는 글리콜 알데히드 (GLA)로 처리하여 재구성 BM (RBM) 행렬의 비 효소 가교이다. AGE 생성 비 효소 적 가교 결합을 확인하는데 사용 GLA의 강성을 증가시킬 수 실험실 기술로서는 RBM이 설명되어 처리 하였다. 광도 분석 및 면역 형광 현미경, 소듐 도데 실 설페이트 폴리 아크릴 아미드에 의한 그것의 비 효소 가교의 AGE 함량 : 이러한 결정 용 (GLA 처리) 단단한 RBM (포스페이트 - 완충 염수, PBS 처리) 네이티브 RBM의 제조를 포함겔 전기 영동 (SDS 페이지)과 공 초점 현미경, 그 증가 된 강성 사용 유변학. 여기에 설명 된 절차는 병든 인간 전립선 조직에 비해 건강에 대한 측정과 일치 3.2 배까지 RBM의 강성 (탄성 계수, E)을 증가시키기 위해 사용될 수있다. 노화와 질병 전립선과 관련된 생물 물리학 적 미세 환경을 다시 만들려면 세 가지 전립선 세포 유형을 기본 RBM과 뻣뻣한 RBM에에 소개 된 글 랜드 : RWPE-1, 전립선 상피 세포 (농도 (PEC)) 정상 전립선에서 파생 된; BPH-1, 양성 전립선 비대증 (BPH)에 의해 영향을받는 전립선 유래의 농도 (PEC); 및 PC3 전이성 세포들은 전립선 암 유래 이차 골 종양으로부터 유래. 여러 매개 변수의 크기, 형상 및 강성 RBM 대 네이티브에 RWPE-1 BPH-1에 의해 형성된 3 차원 샘 꽈리의 침입 특성, 및 평균 셀 길이 이동성 속도 및 3D spher의 세포 이동의 지속성을 포함한 측정 할 수있다동일한 조건 하에서 PC3 세포에 의해 형성 된 OID. 경로 및 단백질의 세포 내 지역화 신​​호 셀도 평가 될 수있다.

뻣뻣한 RBM에 3D 전립선 선포 세포 배양 문화 6 일 후, 농도 (PEC)이 정상 전립선 조직에서 파생 된 (RWPE-1) (그림 1A)와 BPH 조직 (BPH-1) 상피의 균일 한 회전 타원체로 구성됩니다 (그림 1B) 기본 (PBS 치료) RBM에 양식 꽈리 세포. 이 꽈리는 극성 - 기초 - 정점 및 가시 내강 공간 13, 20 고도로 조직 된 페치 특성을 가지고있다. 정상 전립선 조직에서 파생 된 농도 (PEC)에 의해 형성된 꽈리 (RWPE-1) (그림 1A) 및 (GLA 처리) 보강 (AGE 리치) RBM에 BPH 조직 (BPH-1) (그림 1B)를 중단 구조를 가지고 (이동 모양과 세포의 다각형에 구상에서) 나이가 풍부한 RBM) (그림 1A로 선포 세포에서 이주 / 돌출. 이들<html> 꽈리 또한 작거나 존재하지 않는 공간 (13) 내강 그들의 정점 간 기저 극성을 잃었다 고 무질서 농도 (PEC)을 특징으로한다. <img alt="그림 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> 그림 1 :. 기본 및 뻣뻣한 재구성 지하 막 (RBM)에 3D 샘 꽈리로 자란 전립선 상피 세포 (A) RWPE-1 세포의 브라이트 이미지는 PBS (기본) 또는 처리 RBM 젤 6 일 12 시간까지 재배 14 시간 (; 뻣뻣한 AGE 리치) 50 MM의 글리콜 알데히드; 스케일 바는 50 μm의 =. 패널 (A)에 기재된 바와 같이 재배 (B) BPH-1 세포; 스케일 바는 50 μm의 =; 데이터는 3 독립적 인 실험의 대표입니다. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <html> 표 1 &quot;SRC =&quot;/ 파일 / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; 표 1. 기본, 세미 뻣뻣한과 까다로운 재구성 지하 막 (RBM)에 재배 전립선 상피 RWPE-1 꽈리의 특성 RWPE-1 꽈리 GLA (RBM 14 시간 (기본), 글리콜 알데히드 PBS로 전처리 성장했다 ) 6 시간 동안 (반 강성) 또는 14 시간 (딱딱한)에 대한 GLA. 선포 모양의 경우, 백분율 (%)은 라운드의 표준 편차 (SD)를 ±, 반 다각형과 다각형 꽈리는 (50 꽈리는 조건에 따라 정량) 5 독립적 인 실험에서 계산 하였다. 상대 선포 크기는 세 독립적 인 실험에서 (기본 RBM = 100 %)을 산출 하였다. 침입 들어, 하나 이상의 돌출 세포 SD 된 꽈리 % ± 3 독립적 인 실험으로부터 계산 하였다. 배 변화 기본 조건에 대응하는 값에 의해, 반 강성 또는 강성 조건에서 얻어진 평균값으로 나누어 계산한다. P 값은 스튜던트 t 검정 (α = 0.05)을 사용하여 계산 하였다. ontent &quot;FO : 유지-together.within 페이지 =&quot;1 &quot;&gt; AGE 따라 증가 RBM 강성은 PC3 전립선 종양 세포의 이동을 촉진 PC3 세포는 나이가 풍부한 (딱딱한)에 성장하는 반면 기본 RBM 성장 PC3 세포는 서로 (그림 2A)에서 독립적으로 연속 세포 - 세포 접촉을 유지하여 RBM 움직임을 마이그레이션 할 수 있습니다. 문화 PC3 세포에 72 시간은 기본에 초점 (타원체)를 형성 한 후 회전 타원체에서하지 않고 독립적으로 (그림 2B)를 마이그레이션 강성 (AGE 리치) RBM에 PC3 세포 반면, RBM을 (PBS 치료). 뻣뻣한 (AGE 리치) RBM에 PC3 세포는 기본 RBM (그림 2C)에 성장 PC3 세포보다 더 긴합니다. 뻣뻣한 RBM에 PC3 세포는 기본 RBM (그림 2D)에 성장 PC3 세포보다 더 빠르게 이동한다. 뻣뻣한 RBM에 PC3 세포는 네이티브 RBM (도 2e) 상에 성장 PC3 세포에 비해 지속성의 저하를 표시. <img aLT = &quot;그림 2&quot;SRC = &quot;/ 파일 / ftp_upload / 54230 / 54230fig2.jpg&quot;/&gt; 그림 2 :. 기본 및 뻣뻣한 재구성 지하 막 (RBM)에 전립선 종양 세포 마이그레이션 (A) 14 시간 동안 PBS (기본) 또는 50 밀리미터 글리콜 알데히드로 처리 RBM 젤에 성장 PC3 세포의 브라이트 이미지 (AGE-풍부하고 뻣뻣한) . 세포를 명 시야 현미경 (10X 대물 렌즈) 및 궤도를 생성하는 트래킹 셀 다음 12 시간 동안 시간 당 1 화상의 취득 속도를 사용하여 영상화되었다. 도시 된 이미지는 0, 3, 6, 9 및 12 시간 이후의 시점에 대응한다. 단일 세포의 궤도는 12 시간 시점에 대해 표시됩니다. 스케일 바 = 100 μm의. 패널 (A)에 기재된 바와 같이 (B) PC3 세포는 72 시간 동안 천연 또는 뻣뻣한 RBM상에서 배양하고, 묘화. 스케일 바 = 100 μm의. 세부 배의 배율에서 선택한 영역을 보여줍니다. (C) 평균 ± SD 셀 길이 (μm의); 기본 RBM과 뻣뻣한 RBM 사이에 유의 한 차이 (p = 1.2 × 10 -23). ( <str셀 궤도 계산 옹&gt; D) 평균 ± SD 속도 (μm의 / 시간) 기본 RBM과 뻣뻣한 RBM (P = 0.004) 사이의 유의 한 차이. 세포 이동 (E) 평균 ± SD 지속성 (비 D / T, 여기서 D = 처음부터 셀 궤도, 세포 궤도의 T는 = 총 길이의 끝 부분까지의 거리) 기본 RBM과 뻣뻣한 RBM (P = 0.0007) 사이에 유의 한 차이. CE&gt; 10 세포를 분석 하였다 패널의 경우, 데이터가 3 독립적 인 실험의 대표입니다. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

여기에서 우리는 가교 및 당화 산물 (은 AGEs)에 의해 유도 된 기저막 (BM)의 증가 강성 병리학 적 관련성이있는 생물 물리학 적 미세 환경을 모델링하기위한 프로토콜을 설명합니다.

전립선 암과 기타 노화 관련 병리 또는 대사 질환의 생물 물리학 트리거로 지하실 막 강성을 공부하는 방법

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

Research

JoVE Journal

Cancer Research

9.6K 조회수.  Universidad de la República (UDELAR).  여기에서 우리는 가교 및 당화 산물 (은 AGEs)에 의해 유도 된 기저막 (BM)의 증가 강성 병리학 적 관련성이있는 생물 물리학 적 미세 환경을 모델링하기위한 프로토콜을 설명합니다. 

비디오: 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.

공동 배양 시험 법은 아교 모세포종 침략의 대 식세포와 미세 아교 세포 자극을 공부하기

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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암 연구학

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.

그만큼<em&gt; 초파리</em&gt; 가상적인 디스크 종양 모델 : 유전 모자이크를 사용하여 유전자 발현 및 종양 침윤의 시각화 및 정량화

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.

과분극<sup&gt; (13)</sup&gt; C 대사 자기 공명 분광학 및 이미징

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.

콜론-26 암 종양 베어링 암 악액질의 연구를위한 모델로 마우스를

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.

CLIC4 최저 유도 HN4 세포 Apoptosis에서 체 외에 미토 콘 드리 아 막 잠재력의 발견

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.

전립선 암에 있는 Phosphoproteome를 조사 하기 위해 양적 라벨 무료 질량 분석으로 결합 된 Phosphopeptide 농축

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.

인간 P53을 연구하기 위한 기능성 어세이 개발을 위한 섀시로 효모

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.

화학요법 유도점막염의 마우스 모형을 사용하여 작은 장 상해 그리고 적응을 평가하기 위하여 중요한 종점 및 증식 마커

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.

종양 대식 세포 상호 작용을 연구 하는 시험관 내 분석

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.

전립선 암과 기타 노화 관련 병리 또는 대사 질환의 생물 물리학 트리거로 지하실 막 강성을 공부하는 방법

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