Fluorescence-Activated Cell Sorting (FACS) and Molecular Cytogenetics (FISH)

荧光激活细胞分选 (FACS) 和分子细胞遗传学 (FISH)

• 批准号: 7270271
• 负责人: MICHAEL ANDREEFF
• 金额: $ 14.64万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7270271
• 关键词: 5-(6)-carboxyfluorescein diacetate succinimidyl ester; ABCB1 gene; AMD3100; Aftercare; Annexins; Antibodies; Antigens; Apoptosis; Apoptosis Regulator; Apoptotic; Arts; BAY 43-9006; BIRC4 gene; Binding; Biochemical; Biological Assay; Bromodeoxyuridine; CD34 gene; CXCR4 gene; Cell Cycle; Cell Cycle Kinetics; Cell Cycle Stage; Cell Separation; Cell division; Cells; Chromosomes; Cleaved cell; Clinical; Clinical Trials; Clonality; Color; Complement; Confocal Microscopy; DNA Damage; DNA Nucleotidylexotransferase; DNA Probes; DNTT gene; Deposition; Detection; Diploidy; Dissection; Doctor of Medicine; Doctor of Philosophy; Dyes; Event; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Fluorescent in Situ Hybridization; Gene Expression; Genes; Goals; HOE 33342; Hematopoietic; Image Analysis; Immunophenotyping; In Situ Hybridization; Interphase; Interphase Cell; Label; Laboratories; Laser Scanning Cytometry; Lasers; Leukemic Cell; Leukemic Hematopoietic Stem Cell; Localized; MDM2 gene; MDM2 gene; Measurement; Measures; Membrane Lipids; Membrane Potentials; Messenger RNA; Metaphase; Molecular Analysis; Molecular Cytogenetics; Normal Cell; Numbers; PKH 26; Phase; Phenotype; Ploidies; Population; Protein Array; Proteins; Recording of previous events; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Serine; Signal Transduction; Slide; Sorting - Cell Movement; Stem cells; Surface Antigens; System; TP53 gene; Techniques; Testing; annexin A5; base; caspase-3; cytochrome c; human AMID protein; inhibitor/antagonist; lipid structure; mimetics; mitochondrial membrane; novel; progenitor; programs; receptor; red dye CMXRos; response; single cell analysis; stem; survivin; tool; translational study

The FACS/FISH/Confocal Microscopy Core provides functional, phenotypic, cell cycle, genotypic and structural analysis to the investigators of this program project. The laboratory has developed cutting edge techniques in single cell analysis. For the quantitation of apoptotic cells, the TdT-b-dUTP assay (TUNEL) was modified for multiparameter analysis in combination with DMA and BUdR measurements (cell cycle, ploidy). Also, phenotypic analysis of stem and progenitor cells (Project 1), analysis of intracellular proteins related to apoptosis (p53, MDM2, bcl-2, bax, XIAP) (Project 1), and of cell surface antigens including fas and MDR1 is provided. Expression of new regulators of apoptosis proteins (Projects 1, 2) will be analyzed in normal and leukemic cells. Binding of Annexin V to phosphatidyl serine (PS) is utilized for testing changes in the membrane lipid structure associated with apoptosis. A newly developed assay identifies apoptotic cells (PS/Annexin V+) in the AML stem cell compartment (CD34+38"123*). New assays to measure changes in the mitochondrial membrane potential and of cytochrome c in cells initiating apoptosis (CMXRos) and the detection of cleaved caspase 3 are conducted for Projects 1,2,3 and 4. Quantitation of cellular antigens allows us to determine the Antibody Binding Capacity (ABC). CD34 cells are MACS separated for subsequent analysis by FACS. Cell kinetic changes are determined by DNA/Ki67/ CD34/CD38 FCM and the number of actual cell divisions is determined by PKH26 labeling, with cells undergoing none or up to ten divisions being separated by FACS for subsequent molecular analysis (Project 1). Fluorescence in situ hybridization (FISH) determines the number of clonal leukemic interphase or metaphase cells (Projects 4, 5) before and after treatment with the CXCR4 inhibitor AMD3100. The combination of FISH and TUNEL or PS/Annexin V assays allows us to discriminate apoptosis in normal and leukemic cells (Projects 1 and 3) in mixed cell populations. Finally, progenitor and stem cell compartments (CD34+38~, CD34 38~123+ and CD34" lin" cells that eliminate Hoechst 33342, so-called "SP" cells) are sorted for determination of mRNA and protein levels by RPPA. Projects 1 and 2, are analyzed for the presence of clonal leukemic and normal cells. Many of the clinical trial translational studies (BH3 mimetic GX015-070MS, XIAP antisense AEG35156, Raf inhibitor BAY 43-9006 and CXCR4 inhibitor AMD3100 in Projects 4 and 5 are being conducted in Core B1. In addition to multiparametric flow cytometry and cell sorting, the Core provides Laser Scanning Cytometry for quantitation of antigen/proteins in single cells. Importantly, laser confocal microscopy has become an indispensable tool for analysis of intracellular localizations of many proteins, e.g. p53 and AIF (Projects 1,2). The Core provides critical support for all Projects.

FACS/FISH/共聚焦显微镜核心提供功能，表型，细胞周期，基因型和 向该项目的研究人员进行结构分析。该实验室已经开发出尖端的 单细胞分析技术。对于凋亡细胞的定量，TdT-b-dUTP测定（TUNEL） 修改为结合DMA和BUdR测量的多参数分析（细胞周期， 倍性）。此外，干细胞和祖细胞的表型分析（项目1），细胞内蛋白质的分析 与细胞凋亡相关（p53、MDM 2、bcl-2、bax、XIAP）（项目1），以及细胞表面抗原，包括Fas和 提供了MDR 1。新的凋亡蛋白调节因子（项目1，2）的表达将在 正常和白血病细胞。利用膜联蛋白V与磷脂酰丝氨酸（PS）的结合来测试膜联蛋白V的变化。 与凋亡相关的膜脂质结构。一种新开发的检测方法可以识别凋亡细胞 (PS/膜联蛋白V+）在AML干细胞区室（CD 34 +38 - 123*）中。新的检测方法来测量 线粒体膜电位和细胞色素c在细胞启动凋亡（CMXRos）和 对项目1、2、3和4进行了切割的caspase 3检测。细胞抗原定量 抗体结合能力（ABC）。CD 34细胞是MACS分离的， 随后通过FACS进行分析。通过DNA/Ki 67/CD 34/CD 38 FCM和流式细胞仪测定细胞动力学变化。 通过PKH 26标记确定实际细胞分裂的次数，其中细胞不经历分裂或至多经历10次分裂 通过FACS分离分裂以用于随后的分子分析（项目1）。荧光原位 杂交（FISH）确定克隆白血病间期或中期细胞的数量（项目4，5） 在用CXCR 4抑制剂AMD 3100治疗之前和之后。FISH和TUNEL的组合，或 PS/膜联蛋白V测定使我们能够区分正常和白血病细胞的凋亡（项目1和3）， 混合细胞群。最后，将祖细胞和干细胞区室（CD 34 +38-、CD 34 + 38 - 123+和CD 34 - 123+）分成三部分： 分选消除Hoechst 33342的“lin”细胞，所谓的“SP”细胞）用于测定mRNA， 蛋白质水平RPPA分析项目1和2中克隆性白血病细胞和正常细胞的存在。 许多临床试验转化研究（BH 3模拟物GX 015 - 070 MS、XIAP反义AEG 35156、Raf 项目4和项目5中的BAY 43-9006抑制剂和CXCR 4抑制剂AMD 3100正在岩心B1中进行。 除了多参数流式细胞术和细胞分选，Core还提供激光扫描细胞仪 用于定量单细胞中的抗原/蛋白质。重要的是，激光共聚焦显微镜已成为一种 这是分析许多蛋白质（如p53和AIF）细胞内定位的不可或缺的工具（项目1，2）。 核心为所有项目提供关键支持。