CCR, LGI, Flow Cytometry Core Facility

CCR、LGI、流式细胞术核心设施

• 批准号: 8938481
• 负责人: Karen Wolcott
• 金额: $ 90.04万
• 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/8938481
• 关键词: Aerosols; Air Purifying Respirators; Antigens; Antineoplastic Agents; Apoptosis; Appointment; Area; Award; Biological Assay; Breast; CCR; Calendar; Cancer Biology; Cell Death; Cell Line; Cell Separation; Cell fusion; Cell membrane; Cells; Cellular biology; Cherry - dietary; Color; Computer software; Computers; Core Facility; Custom; Cyan Fluorescent Protein; DNA; Data Files; Detection; Doctor of Philosophy; Education; Ensure; Flow Cytometry; Fluorescence-Activated Cell Sorting; Fluorochrome; Funding; Generations; Genes; Genetic; Genome; Goals; Guidelines; Hand; Hepatic; Housing; Human; Human Cell Line; Image; Immune; Immunologist; Institutes; Label; Laboratories; Lasers; Legal patent; Link; Luciferases; Membrane; Mitotic Cell Cycle; Monoclonal Antibodies; Mus; Neoplasm Metastasis; Ovarian; Pancreatic carcinoma; Policies; Process; Proteins; Publications; Questionnaires; RNA; Reporter; Research Personnel; Safety; Sampling; Schedule; Scientist; Services; Signaling Protein; Small Interfering RNA; Sorting - Cell Movement; Staining method; Stains; Subfamily lentivirinae; Supporting Cell; System; Techniques; Thyroid carcinoma; Training; Transfection; Tube; Tumor Cell Line; United States National Institutes of Health; Western Blotting; Work; base; cancer cell; cancer genetics; cancer immunotherapy; cancer stem cell; cell analyzer; design; detector; established cell line; experience; high efficiency particulate air filter; instrument; instrumentation; interest; lung Carcinoma; meetings; nanoparticle; preclinical evaluation; red fluorescent protein; square foot; stable cell line; tumor; vaccine development; web site

The NCI, CCR, LGI Flow Cytometry Core Facility has provided service to 83 Principle Investigators and 236 users from 31 NCI labs or branches this year. The major focus of NCI investigators using the Flow Cytometry Core Facility is to identify and study cancer cells. 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 BD LSRII, BD LSRFortessa flow cytometer cell analyzers, the special order BD FACSAria, and the BC MoFlo Astrios cell sorters 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 facility. The Core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with green, yellow, blue or 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 been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of expression. 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. Last year the Flow Cytometry Core Facility implemented the new policy for cell sorting. According to the 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 was 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 BD FACSAria Fusion cell sorter has been ordered for the purpose of providing this same level of safety when sorting samples of this type. A Class II type A2 Biosafety Cabinet is integrated into the instrument to ensure that aerosols are always contained and evacuated into the HEPA filters. Both cell sorters will be housed in the newly renovated space that is designated for sorts requiring "BSL-2 with Enhanced Precautions". In addition to providing superior biosafety protection the MoFlo Astrios and FACSAria Fusion each have 5 lasers and 18 detectors capable of sorting cells stained with various combinations of fluorochromes. Cells can be sorted as single cells into 96-well plates or bulk sorting into tubes. When additional sorts require "BSL-2 with Enhanced Precautions", Core staff restrict entry to the facility and use powered air purifying respirators (PAPR's) to preform sorts of this kind. Beckman Coulter upgraded the MoFlo Astrios to enable the detection of nano particles. Several investigators are interested in analyzing and sorting exosomes. This is an application that will be developed over the next year. Appointments to use the analyzers are scheduled with shared Microsoft Outlook calendars. The cell sorters are scheduled after the NCI, CCR, LGI 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.

NCI、CCR、LGI 流式细胞术核心设施今年已为来自 31 个 NCI 实验室或分支机构的 83 名主要研究人员和 236 名用户提供了服务。 NCI 研究人员使用流式细胞术核心设施的主要重点是识别和研究癌细胞。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。 BD LSRII、BD LSRFortessa 流式细胞仪细胞分析仪、特殊订单 BD FACSAria 和 BC MoFlo Astrios 细胞分选仪经常用于这些测定。使用表达荧光报告基因的基因转染细胞是大多数使用该设施的实验室所使用的技术。 Core 细胞仪和细胞分选仪配备了特定的激光器，可以检测和分选用绿色、黄色、蓝色或红色荧光蛋白或这些荧光报告基因组合标记的细胞。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。根据绿色或红色荧光蛋白的表达对肿瘤细胞系进行分类，以建立具有高水平表达的细胞系。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。去年，流式细胞术核心设施实施了细胞分选的新政策。根据 NIH 细胞分选仪生物安全政策（2012 年 7 月 28 日），对人体细胞系、人体细胞和人体细胞中的第二代或第三代慢病毒进行分选需要在具有增强预防措施生物安全级别的 BSL-2 上进行分选。 Beckman Coulter MoFlo Astrios 细胞分选机采用定制设计的 SterilGARD II 级生物安全柜进行了升级，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 BD FACSAria Fusion 细胞分选机，以便在分选此类样品时提供相同水平的安全性。仪器中集成了 II 类 A2 型生物安全柜，以确保始终包含气溶胶并将其排入 HEPA 过滤器中。两台细胞分选机都将安置在新装修的空间中，该空间专门用于需要“增强预防措施的 BSL-2”的分选。除了提供卓越的生物安全保护外，MoFlo Astrios 和 FACSAria Fusion 均配有 5 个激光器和 18 个探测器，能够对用各种荧光染料组合染色的细胞进行分选。细胞可以作为单细胞分选到 96 孔板中，也可以批量分选到试管中。当其他类型需要“加强预防措施的 BSL-2”时，核心工作人员会限制进入设施并使用动力空气净化呼吸器 (PAPR) 来执行此类类型的操作。 Beckman Coulter 对 MoFlo Astrios 进行了升级，使其能够检测纳米颗粒。一些研究人员对分析和分选外泌体感兴趣。这是一个将在明年开发的应用程序。使用分析器的约会是通过共享的 Microsoft Outlook 日历安排的。在 NCI、CCR、LGI 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。