R35 Equipment Supplement

摘要 脓毒症、烧伤和创伤是影响我们成年人的最灾难性和最棘手的挑战。 人口创伤、败血症和危重病都是骨髓疾病，我们现在已经证明了 许多长期并发症和令人沮丧的临床结果是继发于“病理激活” 骨髓细胞的数量。我们是第一个报道脓毒症中MDSC亚群扩增的人， 对我们理解这些细胞群有重要贡献。我们在此进行的研究 资助工具已经证明，我们对MDSC表型的定义过于简单，并限制了 治疗MDSC介导的疾病。我们认识到，我们正在进行的研究需要更细粒度的 评估单个干细胞以及骨髓和淋巴细胞的功能和表型 脓毒症、创伤和烧伤后患者的不同结局。为了实现这一目标， 我们需要提高分辨和分离单个MDSC亚型的能力。我们目前正在使用 Bio-Rad S3荧光激活细胞分选仪，已有7年历史，拥有2015年的技术。这包括 只有两个激光器，四种颜色和两个侧流分裂。S3的经验表明， 限制为大约20，000滴/秒和1000事件/秒，使得排序为一天的过程。更 问题，Bio-Rad已经通知我们，他们将停止对S3的支持，包括零件。取而代之， 我们请求支持购买Becton-Dickinson FACSMelody® BRV 9彩色4路流量计 流式细胞仪套装，包括软件、培训和一年的维护合同。的优势 FACSMelody®在现有仪器上的优势非常显著：三个激光器而不是两个激光器，九种颜色而不是四种 颜色和四个与两个侧流同时隔离至少两个和可能三个高得多 MDSC的富集子集。排序速度也很重要。使用目前的仪器， 每个样本需要三到四个小时，而使用新仪器，这些时间可以减少五倍。这不是 不仅增加了通量，而且提高了分离细胞的活力，这是提高严谨性的关键组成部分， 再现性 我们预计每年分析超过75名受试者的样本（150-200个样本），基于 提出的目标。外科系/医学院保证的额外机构资源 服务合同（每年约2.5万美元）是否会在赠款的期限内以及额外试剂的费用 要求更高的特异性（见所附信件）。通过国家地理信息系统和 外科系和医学院，程序目标可以实现高度严谨， 再现性

ABSTRACT Sepsis, burns and trauma are the most catastrophic and intractable challenges affecting our adult population. Trauma, sepsis, and critical illness are diseases of the bone marrow, and we have now demonstrated that many of the long-term complications and dismal clinical outcomes are secondary to ‘pathologic activation’ of myeloid populations. We were the first to report the expansion of MDSC subsets in sepsis, and have made significant contributions to our understanding of these cell populations. Studies we have conducted by this funding instrument have demonstrated that our definitions of MDSC phenotypes over-simplify and limit efforts to prognose and treat MDSC-mediated disease. We recognize that our ongoing studies require a more granular assessment of the function and phenotype of individual stem cell as well as myeloid and lymphoid cell populations over time in patients with differential outcomes after sepsis, trauma and burns. To achieve that goal, we require an increase in our capability to resolve and isolate individual MDSC subtypes. We currently are using a Bio-Rad S3 fluorescently-activated cell sorter that is seven years old, and has 2015 technology. This includes just two lasers, four colors and two side stream splitting. Our experience with the S3 has shown that we are limited to approximately 20,000 drops/second and 1000 events/second making sorts a day-long procedure. More problematic, Bio-Rad has informed us that they are discontinuing support for the S3 including parts. In its stead, we are requesting support for the purchase of a Becton-Dickinson FACSMelody® BRV 9 Color 4 way flow cytometer package including software, training and a one year maintenance contract. The advantages of the FACSMelody® over the existing instrument are significant: three versus two lasers, nine colors instead of four colors and four versus two side streams to simultaneously isolate at least two and likely three much higher enriched subsets of MDSCs. Also important is sorting speed. Using the current instrument, whole blood sorts take three to four hours per sample, and these times can be reduced five-fold with the new instrument. This not only increases throughput, but improves the viability of the isolated cells, a key component of improved rigor and reproducibility. We envision analyzing samples from over 75 subjects annually (150-200 samples) based on the proposed goals. Additional institutional resources guaranteed by the Department of Surgery/College of Medicine will the service contract (~$25k annually) for the life of the grant award and the costs of additional reagents required for the higher specificity (see attached letters). Through combined support from NIGMS and the Department of Surgery and College of Medicine, the programmatic goals can be achieved with high rigor and reproducibility.