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

项目摘要 极晚期抗原-4（VLA-4）细胞表现出独特的行为。与细胞粘附家族中的其他细胞不同， VLA-4通过由内而外的信号传导表现出“滚动和牢固粘附”功能，而同类中的其他细胞 逮捕和坚持。异源二聚体整合素α4-β1可以通过快速的构象变化而发生多种构象变化。 在配体存在下的亲和力调节。还有一个未解决的问题， 这些细胞表面上所有整合素的不一致活化。切线相关，表型免疫 细胞需要使用许多生物标志物来检测分化蛋白质簇的存在， 正常和异常的功能。当追踪肿瘤的进展时，所有生物标志物的检测是至关重要的。 疾病和癌症。这可能会引入高度的复杂性，并且可能变得成本过高 早期的研究者。我们假设1）多谐波高通量流式细胞术将解决多个 分析的每个单细胞的荧光寿命和2）人外周血的表型 使用至少五（5）种生物标志物，用单一激发源激发单核细胞，并建立 一个概念框架的扩展，这种能力，以十个或更多的颜色，而使用单一的激励 源头在相移最大的谐波处采集LDV-FITC荧光寿命。 检测每个细胞的多个荧光寿命将允许研究单个细胞的区域， 以及这种激活是否是细胞的固有区域，或者是否是区域性整合素的激活。 整合素是随机的。通过多参数表型分析可以很好地跟踪疾病和癌症的进展 通过使用多种生物标志物。较新的细胞仪使研究人员能够非常灵敏地检测多种 使用大量颜色通道的生物标记。然而，这引入了仪器化的复杂性 和非常大的数据文件，并且对许多人来说成本过高。根据研究金培训计划，我们将 （2）明确两个目标。我们将1）扩展现有的流式细胞仪，以允许高灵敏度的多谐波 荧光寿命测量和量化每个细胞的整联蛋白行为，和2）使用多寿命 在多个光谱通道上进行表型分析以降低生物仪器复杂性和成本的方法 降低了数据采集和分析的复杂度。

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.