이 작품은 국방부 보조금 W81XWH-14-1-0091 (KMS), Bertarelli Foundation (KMS), 국방부 보조금 R01DC015824 (KMS) 및 T32DC00038 (JES 및 SD 지원) , Nancy Sayles Day Foundation (KMS), Lauer Tinnitus Research Center (KMS) 및 Barnes Foundation (KMS)이 포함됩니다.

<ol>
	<li>Babu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+schwannomas+in+the+modern+era:+epidemiology,+treatment+trends,+and+disparities+in+management.">Vestibular schwannomas in the modern era: epidemiology, treatment trends, and disparities in management.</a> J Neurosurg. 119 (1), 121-130 (2013).</li><li>Gal, T. J., Shinn, J., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+epidemiology+and+management+trends+in+acoustic+neuroma.">Current epidemiology and management trends in acoustic neuroma.</a> Otolaryngol Head Neck Surg. 142 (5), 677-681 (2010).</li><li>Propp, J. M., McCarthy, B. J., Davis, F. G., Preston-Martin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descriptive+epidemiology+of+vestibular+schwannomas.">Descriptive epidemiology of vestibular schwannomas.</a> Neuro Oncol. 8 (1), 1-11 (2006).</li><li>Stangerup, S. E., Tos, M., Thomsen, J., Caye-Thomasen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=True+incidence+of+vestibular+schwannoma?.">True incidence of vestibular schwannoma?.</a> Neurosurgery. 67 (5), 1335-1340 (2010).</li><li>Tos, M., Stangerup, S. E., Caye-Thomasen, P., Tos, T., Thomsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+real+incidence+of+vestibular+schwannoma?.">What is the real incidence of vestibular schwannoma?.</a> Arch Otolaryngol Head Neck Surg. 130 (2), 216-220 (2004).</li><li>Stangerup, S. E., Caye-Thomasen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+natural+history+of+vestibular+schwannomas.">Epidemiology and natural history of vestibular schwannomas.</a> Otolaryngol Clin North Am. 45 (2), 257-268 (2012).</li><li>Mahaley, M. S., Mettlin, C., Natarajan, N., Laws, E. R., Peace, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+patterns+of+care+of+brain+tumor+patients+in+the+United+States:+a+study+of+the+Brain+Tumor+Section+of+the+AANS+and+the+CNS+and+the+Commission+on+Cancer+of+the+ACS.">Analysis of patterns of care of brain tumor patients in the United States: a study of the Brain Tumor Section of the AANS and the CNS and the Commission on Cancer of the ACS.</a> Clin Neurosurg. 36, 347-352 (1990).</li><li>Carlson, M. L., Link, M. J., Wanna, G. B., Driscoll, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+sporadic+vestibular+schwannoma.">Management of sporadic vestibular schwannoma.</a> Otolaryngol Clin North Am. 48 (3), 407-422 (2015).</li><li>Dilwali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporadic+vestibular+schwannomas+associated+with+good+hearing+secrete+higher+levels+of+fibroblast+growth+factor+2+than+those+associated+with+poor+hearing+irrespective+of+tumor+size.">Sporadic vestibular schwannomas associated with good hearing secrete higher levels of fibroblast growth factor 2 than those associated with poor hearing irrespective of tumor size.</a> Otol Neurotol. 34 (4), 748-754 (2013).</li><li>Caye-Thomasen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+and+VEGF+receptor-1+concentration+in+vestibular+schwannoma+homogenates+correlates+to+tumor+growth+rate.">VEGF and VEGF receptor-1 concentration in vestibular schwannoma homogenates correlates to tumor growth rate.</a> Otol Neurotol. 26 (1), 98-101 (2005).</li><li>Koutsimpelas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VEGF/VEGF-R+axis+in+sporadic+vestibular+schwannomas+correlates+with+irradiation+and+disease+recurrence.">The VEGF/VEGF-R axis in sporadic vestibular schwannomas correlates with irradiation and disease recurrence.</a> ORL J Otorhinolaryngol Relat Spec. 74 (6), 330-338 (2012).</li><li>Dilwali, S., Roberts, D., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+VEGF-A+and+cMET+signaling+in+human+vestibular+schwannomas+and+schwann+cells.">Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells.</a> Cancer Biol Ther. 16 (1), 170-175 (2015).</li><li>Dilwali, S., Kao, S. Y., Fujita, T., Landegger, L. D., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsteroidal+anti-inflammatory+medications+are+cytostatic+against+human+vestibular+schwannomas.">Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas.</a> Transl Res. 166 (1), 1-11 (2015).</li><li>Dilwali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+validation+of+anti-nuclear+factor-kappa+B+therapy+to+inhibit+human+vestibular+schwannoma+growth.">Preclinical validation of anti-nuclear factor-kappa B therapy to inhibit human vestibular schwannoma growth.</a> Mol Oncol. 9 (7), 1359-1370 (2015).</li><li>Dilwali, S., Landegger, L. D., Soares, V. Y., Deschler, D. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Factors+from+Human+Vestibular+Schwannomas+Can+Cause+Cochlear+Damage.">Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.</a> Sci Rep. 5, 18599 (2015).</li><li>Blakeley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+drug+treatments+for+neurofibromatosis+type+2-associated+vestibular+schwannoma.">Development of drug treatments for neurofibromatosis type 2-associated vestibular schwannoma.</a> Curr Opin Otolaryngol Head Neck Surg. 20 (5), 372-379 (2012).</li><li>Schroeder, R. D., Angelo, L. S., Kurzrock, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF2/merlin+in+hereditary+neurofibromatosis+2+versus+cancer:+biologic+mechanisms+and+clinical+associations.">NF2/merlin in hereditary neurofibromatosis 2 versus cancer: biologic mechanisms and clinical associations.</a> Oncotarget. 5 (1), 67-77 (2014).</li><li>Schularick, N. M., Clark, J. J., Hansen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+human+vestibular+schwannomas.">Primary culture of human vestibular schwannomas.</a> J Vis Exp. (89), (2014).</li><li>Dilwali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+human+Schwann+and+schwannoma+cells:+improved+and+simplified+protocol.">Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.</a> Hear Res. , 25-33 (2014).</li><li>Brown, C. M., Ahmad, Z. K., Ryan, A. F., Doherty, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+expression+in+sporadic+vestibular+schwannomas.">Estrogen receptor expression in sporadic vestibular schwannomas.</a> Otol Neurotol. 32 (1), 158-162 (2011).</li><li>Cioffi, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-21+overexpression+contributes+to+vestibular+schwannoma+cell+proliferation+and+survival.">MicroRNA-21 overexpression contributes to vestibular schwannoma cell proliferation and survival.</a> Otol Neurotol. 31 (9), 1455-1462 (2010).</li><li>Doherty, J. K., Ongkeko, W., Crawley, B., Andalibi, A., Ryan, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB+and+Nrg:+potential+molecular+targets+for+vestibular+schwannoma+pharmacotherapy.">ErbB and Nrg: potential molecular targets for vestibular schwannoma pharmacotherapy.</a> Otol Neurotol. 29 (1), 50-57 (2008).</li><li>Aguiar, P. H., Tatagiba, M., Samii, M., Dankoweit-Timpe, E., Ostertag, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparison+between+the+growth+fraction+of+bilateral+vestibular+schwannomas+in+neurofibromatosis+2+(NF2)+and+unilateral+vestibular+schwannomas+using+the+monoclonal+antibody+MIB+1.">The comparison between the growth fraction of bilateral vestibular schwannomas in neurofibromatosis 2 (NF2) and unilateral vestibular schwannomas using the monoclonal antibody MIB 1.</a> Acta Neurochir (Wien). 134 (1-2), 40-45 (1995).</li><li>Cattoretti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+recombinant+parts+of+the+Ki-67+antigen+(MIB+1+and+MIB+3)+detect+proliferating+cells+in+microwave-processed+formalin-fixed+paraffin+sections.">Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.</a> J Pathol. 168 (4), 357-363 (1992).</li><li>Hung, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+study+of+human+vestibular+nerve+schwannoma+differentiation.">Immunohistochemistry study of human vestibular nerve schwannoma differentiation.</a> Glia. 38 (4), 363-370 (2002).</li><li>Archibald, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+expression+in+vestibular+schwannomas.">B7-H1 expression in vestibular schwannomas.</a> Otol Neurotol. 31 (6), 991-997 (2010).</li><li>Landegger, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Zinn, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Silico+Reconstruction+of+the+Viral+Evolutionary+Lineage+Yields+a+Potent+Gene+Therapy+Vector.">In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.</a> Cell Rep. 12 (6), 1056-1068 (2015).</li><li>Kim, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulforaphane,+a+natural+component+of+broccoli,+inhibits+vestibular+schwannoma+growth+in+vitro+and+in+vivo.">Sulforaphane, a natural component of broccoli, inhibits vestibular schwannoma growth in vitro and in vivo.</a> Sci Rep. 6, 36215 (2016).</li><li>Soares, V. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+derived+from+human+vestibular+schwannomas+associated+with+poor+hearing+damage+cochlear+cells.">Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.</a> Neuro Oncol. 18 (11), 1498-1507 (2016).</li><li>Lysaght, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+of+human+perilymph.">Proteome of human perilymph.</a> J Proteome Res. 10 (9), 3845-3851 (2011).</li><li>Caye-Thomasen, P., Borup, R., Stangerup, S. E., Thomsen, J., Nielsen, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+genes+in+sporadic+vestibular+schwannomas.">Deregulated genes in sporadic vestibular schwannomas.</a> Otol Neurotol. 31 (2), 256-266 (2010).</li><li>Schulz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+nerve+microenvironment+for+schwannoma+development.">The importance of nerve microenvironment for schwannoma development.</a> Acta Neuropathol. 132 (2), 289-307 (2016).</li><li>Torres-Martin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+profiling+in+vestibular+schwannomas+shows+critical+deregulation+of+microRNAs+and+upregulation+in+those+included+in+chromosomal+region+14q32.">Global profiling in vestibular schwannomas shows critical deregulation of microRNAs and upregulation in those included in chromosomal region 14q32.</a> PLoS One. 8 (6), e65868 (2013).</li></ol>

저자는 공개 할 것이 없습니다.

이 원고는 VS 연구를위한 통일 ​​된 방법 론적 틀을 설명하며, 하류 연구 응용을위한 인간 VS와 GAN 표본의 동시 처리를 개괄합니다. VS 연구가 정밀 의학의 시대에 접어 들면서 수많은 연구 질문에 답할 수있는 형태로 동일한 샘플을 준비하면 개별 환자에게 특유한 분자, 세포, 유전 및 proteomic 통찰력을 발견 할 수 있습니다. 인간 Schwann 세포와 VS 배양의 순도는 S100 마커에 양?...

Vestibular schwannomas (VSs)는 10 만명당 1 명꼴로 진단되는 소뇌 뼈의 각도에서 가장 흔한 신 생물입니다. 비 전이성이더라도이 종양은 환자의 삶의 질 1 , 2 , 3 , 4 , 5 , 6에 심각한 영향을 미칩니다. 영향을받는 개인은 일반적으로 난청, 이명 및 청각 충만감을 느낍니다. 종양이 성장함에 따라 증상이 점점 쇠약하게되어 균형 문제, 안면 마비 및 다른 뇌 기능의 손상을 초래합니다. 뇌간 압박으로 인한 생명을 위협하는 합병증 또한 발생할 수 있습니다. 대장 증후군의 관리 옵션은 근본적으로 정적 종양 및 확립 된 방사선 요법 또는 성장하는 종양의 외과 적 절제를 기다리는 것에주의해야합니다 <sup class = &quot;xref&quot;&gt; 8. 연구 제휴 병원에서이 종양을 외과 적으로 제거하면 환자 수술 중에 수집 된 신선한 종양 조직을 수집하고 분석 할 수있는 기회가 제공됩니다. 관상 동맥 우회술 (translabyrinthine resection) 인 VS에 대한 한 가지 구체적인 외과 적 접근법은 귀와 외음부에서 귀중한 인간 감각 상피에 접근 할 수 있습니다. VS는 말초 감각 신경 ( 즉, 전정 신경)에서 발생하기 때문에 VS 관련 관찰을 인간의 위장 신경 (GAN)과 같은 적절한 제어 신경에서 유도 된 것과 비교하는 것이 중요합니다. 건강한 GAN은 뇌하수체 제거 또는 목구멍 파열시 정기적으로 희생되며 건강한 Schwann 세포 생리학을위한 견고한 모델로 사용될 수 있습니다. Sporadic VS의 치료 나 예방을위한 FDA 승인 의약품이 없기 때문에 연구원들이 근본적인 분자 메커니즘을 명료하게 밝혀야합니다치료 목표를 밝힐 질병의 진전. 혈관 내피 성장 인자 (VEGF), 시클로 옥 시게나 제 2 (COX-2), 핵 인자 카파 B (NF-κB), 종양 괴사 인자 알파 (TNF-α) , 표피 성장 인자 수용체 (EGFR) 및 관련 신호 분자 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17을 포함한다 . 최근의 진보는 일차 인간 전정 신경초와 건강한 신경 조직의 수집, 처리, 배양 및 하류 조사를위한 프로토콜을 확장하고 개선했습니다 18 , 19 . 그러나, 대부분의 기존의 프로토콜은 준비를 수용하도록 설계되었습니다단일 하류 연구 응용 ( 즉, 세포 배양 만)에 대한 그러한 조직의 배양. 이 기사는 세포 유형 특이 적 배양, 단백질 추출, RNA 보존, 종양 분비 수집 및 조직 고정과 같은 다중 다운 스트림 응용 분야에 대한 단일 기본 인간 VS 또는 GAN 시료의 동시 처리를위한 통일 ​​된 방법 론적 틀을 제시합니다. 이 작품은 translabyrinthine VS 절제술 동안 인간 뇌척수액 (CSF)과 외음근의 외과 수집 및 가공에 대해 자세히 설명합니다.이 밀접한 관련 조직은 VS에 대한 중요한 바이오 마커의 역할을 할 수 있습니다. 마지막으로,이 프로토콜은 유전자 치료에 사용하기 위해이 조직의 새로운 응용 프로그램으로 문화에서 기본 인간 VS 세포의 바이러스 성 전달을위한 단계를 제공합니다.

<table border="0" cellpadding="0" cellspacing="0" width="1419">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">BioCoat Poly-D-Lysine/Laminin 12mm #1 German Glass Coverslip</td>
			<td style="width:159px;">Corning</td>
			<td style="width:159px;">354087</td>
			<td style="width:683px;">Or prepare coverslips with Corning Laminin (CB-40232) and Cultrex Poly-L-Lysine (3438-100-01)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">188161</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">CELLSTAR Cell Culture Dish, 60 mm</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Collagenase from Clostridium histolyticum, Sterile-filtered</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">C1639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Costar 24 Well Clear TC-Treated Multiple Well Plates, Sterile</td>
			<td style="width:159px;">Corning</td>
			<td style="width:159px;">3526</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">D1306</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">DMEM, high glucose, pyruvate, no glutamine, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">DMEM/F-12, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">11320-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dumont #3 Forceps, Dumoxel</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">11231-30</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dumont #5 Forceps, Standard tip, Inox</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">11251-20</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10437-028&#160;</td>
			<td>Aliquot in 50 ml tubes and store in -20&#176;C freezer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Hyaluronidase from Bovine Testes, Type I-S, Lyophilized Powder</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">H3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Millex-GP Syringe Filter Unit, 0.22 &#181;m, polyethersulfone, 33 mm, sterile</td>
			<td style="width:159px;">EMD Millipore</td>
			<td style="width:159px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Paraformaldehyde, Reagent Grade, Crystalline</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">P6148</td>
			<td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">PBS, pH 7.4, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10010-023&#160;</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Penicillin-Streptomycin (10,000 U/ml), 100 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">PhosSTOP Phosphatase Inhibitor Tablets</td>
			<td style="width:159px;">Roche</td>
			<td style="width:159px;">04906845001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Pierce Protease Inhibitor Tablets</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">88666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Pipettes and pipette tips, 5/10/25 ml</td>
			<td style="width:159px;">Variable</td>
			<td style="width:159px;">Variable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Plastic Homogenization Pestle for 1.5/2.0ml Microtubes</td>
			<td style="width:159px;">E&#38;K Scientific</td>
			<td style="width:159px;">EK-10539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">PrecisionGlide Needles, 27 G x 1 1/2 in&#160;</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">301629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">RIPA Buffer</td>
			<td style="width:159px;">Boston BioProducts</td>
			<td style="width:159px;">BP-115</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">RNAlater (RNA stabilization solution)</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">AM7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:159px;">022363719</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:159px;">022363204</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Saline - 0.9% Sodium Chloride Injection, bacteriostatic, 20 ml</td>
			<td style="width:159px;">Hospira</td>
			<td style="width:159px;">0409-1966-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Scalpel Blades - #15</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">10015-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Schuknecht Suction Tube 24 gauge</td>
			<td style="width:159px;">Bausch + Lomb</td>
			<td style="width:159px;">N1698 42</td>
			<td>Useful for the surgical approach (in addition to common otologic surgical instruments) and e.g. a blue surgical marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Specimen Container, OR sterile, 4OZ&#160;</td>
			<td style="width:159px;">Medline</td>
			<td style="width:159px;">DYND30331H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Stemi 2000-C Stereo Microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:159px;">000000-1106-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Syringe/Needle Combination, Luer-Lok Tip, 5 ml, 22 G x 1 in.</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tuberculin Syringe Only, Slip tip, 1 ml</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tuberculin Syringe Only, Slip tip, 3 ml</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Ultrasonic homogenizer, 4710 Series, CV18 probe</td>
			<td style="width:159px;">Cole-Parmer</td>
			<td style="width:159px;">CP25013</td>
			<td></td>
		</tr>
	</tbody>
</table>

a unified methodological framework for vestibular schwannoma research

전정 신경초종은 cerebellopontine 각의 가장 흔한 신 생물이며 모든 두개 내 성장의 6-8 %를 차지합니다. 이 종양은 영향받은 개체의 최대 95 %에서 감각 신경성 청력 손실을 일으키지 만,이 청력 손실의 근간을 이루는 분자 메커니즘은 아직 파악하기 어렵습니다. 이 기사는 전정 신경초종 연구에 필수적인 다운 스트림 연구 응용을위한 다양한 1 차 인간 조직 샘플의 수집과 처리를 용이하게하기 위해 연구실에서 수립 된 단계를 설명합니다. 특히,이 연구는 외과 표본에서 Schwann과 schwannoma 세포의 수집, 가공 및 배양에 대한 통일 된 방법 론적 틀을 설명합니다. 이것은 종양과 신경 분비물의 수집, RNA의 보존과 수집 된 조직에서의 단백질 추출, 절편 준비를위한 조직 고정, 현재의 연구에 필수적인 것으로 간주되는 병행 처리 단계와 통합되어 있습니다.유전자 치료에 응용하기 위해 아데노 관련 바이러스에 일차 인간 세포를 노출시키는 것. 또한,이 연구는이 종양을 내이 및 외음부에서 인간의 감각 상피를 얻을 수있는 독특한 기회로 수집하는 외과 적 접근법을 강조합니다. 실험적인 품질을 향상시키기위한 팁이 제공되고 일반적인 함정이 강조 표시됩니다.

5 장에서 확립 된 것처럼 배양 된 1 차 인간 VS 세포는 많은 하류 연구 응용에서 질병 관련 과정에 대한 유익한 모델로 취급 될 수있다 ( 그림 1 ). 6 절에서 배양 된 건강한 Schwann 세포는 직접적이고 객관적인 비교 점을 ​​제공합니다. 아래에 요약했듯이이 통일 된 방법론 프레임 워크에 따라 처리 된 VS 및 GAN의 광범위한 데이터는 이전에 <sup class="xref"...

Watch this Scientific Journal Video about A Unified Methodological Framework for Vestibular Schwannoma Research at JoVE.com

A Unified Methodological Framework for Vestibular Schwannoma Research

이 프로토콜의 목표는 vestibular schwannoma 및 Schwann 세포 연구에서 여러 하류 응용 프로그램에 대한 인간의 수술 샘플 수집 및 처리를 설명하는 것입니다.

Vestibular Schwannoma 연구를위한 통합 방법론 체계

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these ...

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Cancer Research

7.3K 조회수.  Massachusetts Eye and Ear.  이 프로토콜의 목표는 vestibular schwannoma 및 Schwann 세포 연구에서 여러 하류 응용 프로그램에 대한 인간의 수술 샘플 수집 및 처리를 설명하는 것입니다. 

비디오: A Unified Methodological Framework for Vestibular Schwannoma Research

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 ...

연구

암 연구학

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

종양의 성장과 전이 연구 추적 가능한 기자와 유방암 환자 유래의 이종 이식의 라벨링

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

E-Patient Counseling Trial (E-PACO): Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and perform a risk assessment for sedative use prior to a colonoscopy. The current practice in the Netherlands to achieve these goals is nurse counseling in an outpatient setting. This is costly and has disadvantages in terms of uniformity and time consumption for both the patient and the hospital. The hypothesis is that computer-based education with use of video and 3D animations may replace nurse counseling in most cases, without losing quality of bowel cleanliness during colonoscopy.
This multicenter, randomized, endoscopist blinded clinical trial evaluates a primary outcome measure (bowel preparation) during colonoscopy. Secondary outcome measures are sickness absence, patient anxiety after instruction and prior to colonoscopy, patient satisfaction and information re-call. The study will be performed in four endoscopy units of different levels (rural, urban, and tertiary). Inclusion criteria are adult age and referral for complete colonoscopy. Exclusion criteria are Dutch illiteracy, audiovisual handicaps or mental disabilities and no (peers with) internet access.
This trial aims to establish online computer-based education as tool for patient education prior to a colonoscopy. By choosing a direct comparison with the standard of care (nurse counseling), both endoscopic quality measures and patient related outcome measures can be evaluated.

E-Patient Counseling Trial E-PACO: Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

The aim of this trial is to establish the position of online computer-based education as a tool for patient preparation prior to a colonoscopy. Computer based education is compared with the standard of care, nurse counseling, evaluating endoscopic quality measures and patient related outcome measures.

전자 환자 상담 재판 (E-PACO): 대장 내시경 검사를 준비하는 환자를위한 컴퓨터 기반 교육 대 간호사 상담

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

생산 및 매일 임상 연습에서 신경 내 분 비 종양에 대 한 68Ga-DOTATATE의 PET/CT 영상에 대 한 실용적인 가이드

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

종양 침투 T 세포 독성의 정형 외 췌장 종양 및 Ex 생체 특성의 생성

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

라이브 셀에서 eIF4E-eIF4G 상호 작용을 측정하여 eIF4F 어셈블리 모니터링

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

교모세포종을 위한 3D 스페로이드 모형

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

인간 결장 암 세포에 있는 DNA 손상 및 복구 Foci의 면역 형광 화상 진찰

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

낮은 종양 함량 조직에서 종양 농축을 위한 자동화된 해부 프로토콜

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...