Pruitt Margaret M, Marin Wilfredo, Waarts Michael R, de Jong Jill L O
Department of Pediatrics, Section of Hematology-Oncology, The University of Chicago; Department of Biology and Biotechnology, Worcester Polytechnic Institute.
Department of Pediatrics, Section of Hematology-Oncology, The University of Chicago.
J Vis Exp. 2017 May 4(123):55711. doi: 10.3791/55711.
Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.
来自健康或恶性组织的异质细胞群体可能包含一群细胞，其特征是对DNA结合染料Hoechst 33342的外排能力存在差异。在用紫外线（UV）激光激发Hoechst 33342染料后，可使用流式细胞术方法识别这群“侧群”细胞。许多细胞类型的侧群包含干细胞或祖细胞样细胞。然而，并非所有细胞类型都有可识别的侧群。斑马鱼（Danio rerio）具有强大的T细胞急性淋巴细胞白血病（T-ALL）体内模型，但这些斑马鱼T-ALL是否有侧群尚不清楚。这里描述的方法概述了如何分离斑马鱼T-ALL中的侧群细胞。首先，通过将tol2质粒显微注射到单细胞期胚胎中来产生斑马鱼的T-ALL。一旦肿瘤生长到扩展到动物身体一半以上的阶段，就可以收获T-ALL细胞。然后用Hoechst 33342对细胞进行染色，并通过流式细胞术检测侧群细胞。该方法在斑马鱼T-ALL研究中有广泛应用。虽然在斑马鱼中没有已知的细胞表面标志物来确认这些侧群细胞是否类似癌症干细胞，但体内功能移植试验是可行的。此外，单细胞转录组学可用于识别这些侧群细胞的遗传特征。
