细胞静止是正常干细胞和癌症干细胞 (CSC) 中描述的一种生长停滞或增殖减慢的状态。静止可以保护 CSC 免受抗增殖化疗药物的影响。在 T 细胞急性淋巴细胞白血病 (T-ALL) 患者来源的异种移植 (PDX) 小鼠模型中，静止细胞与治疗耐药性和干性相关。细胞增殖染料是追踪细胞分裂的流行工具。荧光染料共价固定在膜上的胺基和细胞内的大分子上。这允许跟踪标记细胞最多 10 个分裂，这可以通过流式细胞术来解决。最终，增殖率最高的细胞的染料保留率较低，因为每次细胞分裂都会稀释染料，而休眠、分裂较慢的细胞则具有最高的保留率。使用细胞增殖染料分离休眠细胞已在 T-ALL 小鼠模型中得到优化和描述。作为对现有小鼠模型的补充，rag2:Myc 衍生的斑马鱼 T-ALL 模型为研究 T-ALL 的自我更新提供了绝佳的场所，因为白血病干细胞 (LSC) 的频率很高，而且斑马鱼可以方便地进行大规模研究。 -规模移植实验。在这里，我们描述了用细胞增殖染料对斑马鱼 T-ALL 细胞进行染色的工作流程，优化斑马鱼细胞的染料浓度，在体内成功传代染色的细胞，以及通过以下方法收集具有不同染料保留水平的细胞：对移植动物的活细胞进行分选。鉴于 T-ALL 中缺乏成熟的 LSC 细胞表面标记物，该方法提供了一种在体内询问静止细胞的功能方法。为了获得代表性结果，我们描述了高和低染料保留细胞的植入效率和 LSC 频率。该方法可以帮助研究静止细胞的其他特性，包括药物反应、转录谱和形态。

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry. Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments. Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.