Pronk Cornelis J H, Bryder David
Division of Molecular Hematology, Institution for Laboratory Medicine, Lund University, Klinikgatan 26, 221 84, Lund, Sweden.
Department of Pediatric Oncology/Hematology, Skane University Hospital, 221 85, Lund, Sweden.
Methods Mol Biol. 2018;1678:301-319. doi: 10.1007/978-1-4939-7346-0_13.
Myeloerythroid-restricted precursor cells, derived from multipotent hematopoietic stem cells, give rise to mature cells of the granulocyte, monocyte, erythroid, and/or thrombocytic lineages. High-resolution profiling of the developmental stages, from hematopoietic stem cells to mature progeny, is important to be able to study and understand the underlying mechanisms that guide various cell fate decisions. Also, this approach opens for greater insights into pathogenic events such as leukemia, diseases that are most often characterized by halted differentiation at defined immature precursor levels. In this chapter, we provide protocols and discuss approaches concerning the analysis and purification of immature myeloerythroid lineages by multiparameter flow cytometry. A wealth of literature has demonstrated the feasibility of similar approaches also for the human system. However, in this chapter, we focus on the identification of bone marrow cells derived from C57BL/6 mice, in which flow cytometry-based immunophenotypic applications have been most widely developed. This should allow also for its application in genetically modified models on this background. For maximal reproducibility, all protocols described have been established using reagents from commercial vendors to be analyzed on a flow cytometer with factory standard configuration.
源自多能造血干细胞的髓系红系限制性前体细胞可分化为粒细胞、单核细胞、红细胞和/或血小板谱系的成熟细胞。对从造血干细胞到成熟子代的发育阶段进行高分辨率分析，对于研究和理解指导各种细胞命运决定的潜在机制至关重要。此外，这种方法有助于更深入地了解白血病等致病事件，这些疾病通常以特定未成熟前体水平的分化停滞为特征。在本章中，我们提供了通过多参数流式细胞术分析和纯化未成熟髓系红系谱系的方案并讨论相关方法。大量文献已证明类似方法在人类系统中也具有可行性。然而，在本章中，我们重点关注源自C57BL/6小鼠的骨髓细胞的鉴定，基于流式细胞术的免疫表型分析在此背景下得到了最广泛的发展。这也应使其能够应用于基于此背景的基因编辑模型。为了实现最大程度的可重复性，所有所述方案均使用来自商业供应商的试剂，并在具有工厂标准配置的流式细胞仪上进行分析。
