A high-throughput approach to quantify cellular integrin conformation and cell differentiation using multiharmonic, multicolor fluorescence lifetime-dependent flow cytometry

使用多谐波、多色荧光寿命依赖性流式细胞术量化细胞整合素构象和细胞分化的高通量方法

• 批准号: 9427838
• 负责人: Jesus Salvador Sambrano
• 金额: $ 1.87万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2018-10-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9427838
• 关键词: Address; Affinity; Avidity; Behavior; Biochemical; Biological Markers; Biomedical Research; Blood; Cell Adhesion; Cell Differentiation process; Cell surface; Cells; Color; Complex; Data; Data Analyses; Data Files; Detection; Development; Dimensions; Disease; Disease Progression; Event; Exhibits; Family; Fellowship; Financial compensation; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence; Fluorochrome; Foundations; Future; Goals; Heterogeneity; Human; Immune; Immune response; Immunity; Immunology; Immunophenotyping; Institution; Integrin alpha4; Integrin alpha4beta1; Integrins; Investigation; Lasers; Leukocytes; Ligands; Lipid Bilayers; Malignant Neoplasms; Measurement; Mediating; Molecular Conformation; Nature; Non-Hodgkin' s Lymphoma; Pathogenesis; Pathology; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Population; Process; Proteins; Protocols documentation; Research Personnel; Rest; Short Interspersed Nucleotide Elements; Signal Transduction; Source; Specific qualifier value; Surface; System; Testing; Time; Training; Work; cost; cost efficient; data acquisition; dimer; drug development; fMet-Leu-Phe receptor; immune function; instrument; instrumentation; novel; screening; single cell analysis; tenure track; tool

Project Abstract Very Late Antigen-4 (VLA-4) cells exhibit a unique behavior. Unlike other cells in the cell adhesion family, VLA-4 exhibit a ‘roll and firmly adhere’ function through inside-out signaling where other cells in the same class arrest and adhere. Heterodimeric integrins α4-β1 can undergo several conformational changes through rapid affinity modulation in the presence of ligands. There remains an unresolved issue with incomplete and inconsistent activation of all integrins on the surface of these cells. Tangential related, phenotyping immune cells requires use of many biomarkers to detect the presence of clusters of differentiated proteins as well as their normal and abnormal functions. Detection of all biomarkers is crucial when tracking the progression of diseases and cancers. This may introduce a high level degree of complexity, and may become cost-prohibitive to early investigators. We hypothesize 1) multiharmonic high-throughput flow cytometry will resolve multiple fluorescence lifetimes for each single cell analyzed and 2) phenotyping of human peripheral blood mononuclear cells using a minimum of five(5) biomarkers, excite with a singular excitation source and establish conceptual framework for the expansion of this ability to ten or more colors while use of a singular excitation source. Acquisition of LDV-FITC fluorescence lifetimes at harmonics where phase shift is at a maximum. Detection of multiple fluorescence lifetimes per cell will allow for investigation of regions of a single cell that contain activated integrins and whether this activation is an inherent region of the cell or if activation of regional integrins is random. Progression of diseases and cancer is well tracked through multiparametric phenotyping through use of multiple biomarkers. Newer cytometers allow investigators very sensitive detection of multiple biomarkers using extensive number of color channels. However this introduces complexity in instrumentation and very large data files and becomes cost-prohibitive to many. Under the fellowship training plan, we will address two(2) specific aims. We will 1) expand current flow cytometer to allow highly sensitive multiharmonic fluorescence lifetime measurements and quantify integrin behavior per cell and 2) use the multi-lifetime approach in phenotyping across multiple spectral channels to reduce bioinstrumentation complexity and cost and reduce complexity of data acquisition and analysis.

项目摘要