FACSGuard - An additive for improving flow cytometry cell yield

描述（由申请人提供）：在基础和应用研究中越来越多地使用流式细胞术来完成诊断测定。其中一个研究领域是研究从肿瘤中脱落的循环细胞。另一个非常活跃和当代的领域是产生流式细胞术侧群，其含有多能癌症干细胞，其可以存在于少于1%的细胞群中。此外，众所周知，几乎所有类型的干细胞（包括人胚胎干细胞）的流式细胞术表征都是一个挑战，这是由于分选过程中的高细胞损失。这使得一些人考虑在细胞分选方案中包含ROCK抑制剂，以增加活力并改善干细胞表面标志物的检测。为了应对这些挑战，CPSI Biotech建议开发第一种与FACS兼容的分选试剂，称为FACSGuard，专门设计用于在流式细胞术过程中保护细胞。该培养基将被设计为在常温（37 ℃）下使用，以改善在典型的细胞解离、分离和流动过程中激活的几种应激途径。如果成功，该试剂可用于各种流式细胞术应用，从低应力番石榴到更传统的FACS系统中通常需要的高PSI（70），以分离其他细胞类型。该项目的具体目的如下：（1）在模拟流式细胞术条件下细胞经历的条件下测试各种候选细胞应激途径抑制剂，以可能纳入FACSGuard;（2）开发几种版本的FACSGuard;（3）在两种人胚胎干细胞系以及其他更稳健的细胞类型上测试FACSGuard版本。第2阶段将致力于使用RT-PCR和其他微阵列技术，以确定一种通用FACSGuard试剂是否足以用于大多数流式细胞术应用，或者是否应开发几种细胞和/或条件特异性变体。该项目的成功完成将导致识别和理解在FACS侧群中发现的癌症干细胞的改进方法，以及直接和间接地改进干细胞治疗应用。 公共卫生相关性：该项目旨在为干细胞开发一种保护性解决方案，以便在分离和分析过程中保护其活力和功能。这将直接有益于从理解癌症干细胞到干细胞治疗的各个领域。

DESCRIPTION (provided by applicant): There is increasing utilization of flow cytometry in both basic and applied research to accomplish diagnostic assays. One such area of research is the investigation of circulating cells that have detached from tumors. Another very active and contemporary area is the generation of flow cytometry side populations that contain multipotent cancer stem cells that can be present in less than 1% of the cell population. Furthermore, it is well known that the flow cytometry characterization of nearly all types of stem cells, including human embryonic stem cells, is a challenge due to the high cell loss during the sorting process. This has led some to consider the inclusion of ROCK inhibitors in the cell sorting protocol to increase viability and improve the detection of stem cell surface markers. To address these challenges CPSI Biotech proposes to develop the first FACS compatible sorting reagent, referred to as FACSGuard, expressly designed to protect cells during the flow cytometry process. This media will be designed to be used at normothermic (37oC) temperatures to ameliorate several stress pathways that are activated during a typical cell dissociation, separation and flow procedure. If successful, this reagent can be used in a variety of flow cytometry applications from low stress Guava to the high PSI (70) often required in more traditional FACS systems to separate other cell types. The Specific Aims of the project are the following: (1) Test a variety of candidate cell stress pathway inhibitors in conditions that mimic those experienced by cells under flow cytometry conditions for possible inclusion in FACSGuard; (2) Develop several versions of FACSGuard; and (3) Test the versions of FACSGuard on two human embryonic stem cell lines as well as other more robust cell types. Phase 2 will be dedicated to using RT-PCR and other microarray techniques to determine if one generic FACSGuard reagent is sufficient for most flow cytometry applications or if several cell and or condition-specific variants should be developed. The successful completion of this project will lead to an improved method of identifying and understanding cancer stem cells found in FACS side populations as well as directly and indirectly improve stem cell therapy applications. PUBLIC HEALTH RELEVANCE: This project is designed to develop a protective solution for stem cells so that both their viability and function can be protected during their separation and analysis. This will have direct benefit to diverse areas ranging from understanding cancer stem cells to stem cell therapy.