CCR FACS Core Laboratory

CCR FACS核心实验室

• 批准号: 8763776
• 负责人: Karen Wolcott
• 金额: $ 93.97万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763776
• 关键词: Aerosols; Air Purifying Respirators; Antigens; Antineoplastic Agents; Apoptosis; Appointment; Area; Award; Biological Assay; Breast; CCR; Calendar; Cell Death; Cell Line; Cell Separation; Cell membrane; Cells; Cellular biology; Computer software; Computers; Core Facility; Custom; Cyan Fluorescent Protein; DNA; Data Analyses; Detection; Doctor of Philosophy; Education; Filtration; Flow Cytometry; Fluorescence-Activated Cell Sorting; Funding; Future; Generations; Genes; Guidelines; Hepatic; Hour; Housing; Human; Human Cell Line; Image; Immunologist; Institutes; Label; Laboratories; Lasers; Legal patent; Licensing; Link; Luciferases; Membrane; Mitotic Cell Cycle; Monoclonal Antibodies; Mus; Neoplasm Metastasis; Ovarian; Pancreatic carcinoma; Policies; Proteins; Publications; Questionnaires; RNA; Reporter; Research; Research Personnel; Running; Sampling; Schedule; Scientist; Services; Signaling Protein; Site; Small Interfering RNA; Sorting - Cell Movement; Subfamily lentivirinae; System; Techniques; Thyroid carcinoma; Time; Training; Transfection; Tumor Cell Line; United States National Institutes of Health; Viola; Western Blotting; Work; base; cancer immunotherapy; cancer stem cell; cell analyzer; design; established cell line; experience; instrument; instrumentation; lung Carcinoma; preclinical evaluation; red fluorescent protein; square foot; stable cell line; tumor; vaccine development; web site

The NCI, CCR, LCBG Flow Cytometry Core Facility has provided service to 78 Principle Investigators and 223 users from 31 NCI labs or branches from January to August 2013. The flow cytometry cell analyzers were used a total of 1,803 hours; FACS Caliburs for 387 hours, LSRII for 558 hours, and the LSRFortessa for 858 hours. During the same time, flow cytometry cell sorters were used a total of 1,370 hours; FACSAria-UV for 553 hours, FACSAria-Violet for 636 hours and the MoFlo Astrios for 181 hours. Identifying and studying cancer stem cells are one of the major research areas of the Flow Cytometry Core Facility users. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic and lung carcinomas. The LSRII flow cytometer, the special order FACSAria, and the MoFlo Astrios cell sorter are frequently used for these assays. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of labs using the Flow Cytometry Core Facility. The core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with any of the green, yellow, blue and red fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have then been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of luciferase. These cell lines have then been used to establish tumors in mice and to image metastasis. Flow cytometry is also frequently used for looking at cell growth (cell cycle analysis) and mechanisms of cell death (apoptosis) to examine actions of cancer drugs. The Flow Cytometry Core Facility has implemented a new policy for cell sorting. According to the recently developed NIH Policy for Biosafety of Cell Sorters (July 28, 2012) sorting of human cell lines, human cells and 2nd or 3rd generation Lentivirus in human cells require the sort to be performed at a BSL-2 with enhanced precautions biosafety level. The Beckman Coulter MoFlo Astrios cell sorter has recently been upgraded with a custom designed SterilGARD Class II Biosafety Cabinet with an onboard HEPA filtration system that is uniquely configured to fit on the instrument bench to provide safe, effective aerosol evacuation. This instrument upgrade enables the flow cytometry staff to perform sorts in compliance with the new guidelines. A second Baker Class II Biosafety Cabinet has been ordered and will house a BD FACSAria in the future. Additionally, plans to renovate the facility to provide a separate space for the MoFlo Astrios and a FACSAria so that sorts requiring this biosafety level can be done every day rather than only two times a week. Currently, core staff restrict entry to the facility and use powered air purifying respirators (PAPR's) on Tuesday and Thursday to preform sorts of this kind. Appointments to use the analyzers are scheduled with shared Microsoft Outlook calendars. The cell sorters are scheduled after the NCI, CCR, LCBG Flow Cytometry Core Facility Biosafety Questionnaire and Sort Request form has been submitted and reviewed by the Core Manager. The core website, located at https://labshare.nih.gov/nci/ccr-facs is currently under construction. It will soon contain pertinent information about the services the core offers as well as provide an easy scheduling system.

2013年1月至2013年8月，NCI、CCR、LCBG流式细胞术核心设施已为NCI 31个实验室或分支机构的78名主要研究者和223名用户提供服务。流式细胞分析仪总计使用1,803小时； FACS Caliburs 为 387 小时，LSRII 为 558 小时，LSRFortessa 为 858 小时。在此期间，流式细胞仪细胞分选仪总共使用了1,370小时； FACSAria-UV 为 553 小时，FACSAria-Violet 为 636 小时，MoFlo Astrios 为 181 小时。识别和研究癌症干细胞是流式细胞术核心设施用户的主要研究领域之一。许多 NCI 实验室正在使用流式细胞术和荧光激活细胞分选技术，通过单克隆抗体的膜抗原表达或涉及活性膜底物转运的功能测定来识别和分选癌症干细胞。 CCBB、MBTL、LEC、LHC 和 LCBG 的研究人员正在研究来自乳腺癌、卵巢癌、肝癌、甲状腺癌、胰腺癌和肺癌的癌症干细胞。 LSRII 流式细胞仪、特殊订单的 FACSAria 和 MoFlo Astrios 细胞分选仪经常用于这些测定。用表达荧光报告基因的基因转染细胞是大多数使用流式细胞术核心设施的实验室所使用的技术。核心细胞仪和细胞分选仪配备了特定的激光器，可以检测和分选用任何绿色、黄色、蓝色和红色荧光蛋白或这些荧光报告基因的组合标记的细胞。分选的转染细胞用于制备可用于蛋白质印迹和微阵列的蛋白质、DNA 和 RNA。分选的细胞还用于确定 siRNA 的效果、观察信号蛋白，或者可以进一步传代以创建稳定的细胞系。荧光报告蛋白也可能与荧光素酶连接。然后根据绿色或红色荧光蛋白的表达对肿瘤细胞系进行分类，以建立具有高水平荧光素酶的细胞系。这些细胞系随后被用于在小鼠中建立肿瘤并对转移进行成像。流式细胞术还经常用于观察细胞生长（细胞周期分析）和细胞死亡（细胞凋亡）机制，以检查抗癌药物的作用。流式细胞术核心设施实施了一项新的细胞分选政策。根据最近制定的 NIH 细胞分选仪生物安全政策（2012 年 7 月 28 日），对人类细胞系、人类细胞和人类细胞中的第二代或第三代慢病毒进行分选，需要在 BSL-2 上进行分选，并采取增强的生物安全预防措施等级。 Beckman Coulter MoFlo Astrios 细胞分选机最近进行了升级，配备了定制设计的 SterilGARD II 级生物安全柜，该柜配有板载 HEPA 过滤系统，该系统经过独特配置，适合安装在仪器台上，以提供安全、有效的气溶胶排出。此次仪器升级使流式细胞仪工作人员能够按照新指南进行分类。已订购第二个贝克 II 级生物安全柜，将来将容纳 BD FACSAria。此外，计划翻新该设施，为 MoFlo Astrios 和 FACSAria 提供单独的空间，以便每天都可以进行需要这种生物安全级别的分类，而不是每周只进行两次。目前，核心工作人员限制进入该设施，并在周二和周四使用电动空气净化呼吸器（PAPR）来进行此类活动。使用分析器的约会是通过共享的 Microsoft Outlook 日历安排的。在 NCI、CCR、LCBG 流式细胞术核心设施生物安全问卷和分选申请表提交并由核心经理审核后安排细胞分选。核心网站位于 https://labshare.nih.gov/nci/ccr-facs 目前正在建设中。它将很快包含有关核心提供的服务的相关信息，并提供一个简单的调度系统。