T淋巴细胞中转录因子的流式细胞术分析。
Flow cytometry analysis of transcription factors in T lymphocytes.
作者信息
Albu Diana I, Califano Danielle, Avram Dorina
机构信息
Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY, USA.
出版信息
Methods Mol Biol. 2010;647:377-90. doi: 10.1007/978-1-60761-738-9_23.
Abstract
Detection of transcription factors in immune cell populations, particularly in subpopulations that are represented at low frequencies in lymphoid and nonlymphoid organs, presents a particular challenge when using traditional methods such as western blot analysis. Therefore, development of flow cytometry-based methods which allow identification of transcription factors in specific immune cell populations is of main interest. Here we developed and optimized a methodology for rapid and convenient detection of the transcription factor BCL11B in T lymphocyte subpopulations using flow cytometry. The optimal protocol employs saponin and Tween 20 both during the fixation and permeabilization steps, and we demonstrate that it is efficient for three anti-BCL11B antibodies covering distinctive BCL11B epitopes. In addition, we prove that the method preserves the staining of surface markers.
摘要
在免疫细胞群体中检测转录因子，尤其是在淋巴器官和非淋巴器官中以低频率存在的亚群中检测转录因子时，使用蛋白质印迹分析等传统方法会面临特殊挑战。因此，开发基于流式细胞术的方法以识别特定免疫细胞群体中的转录因子备受关注。在此，我们开发并优化了一种利用流式细胞术快速便捷地检测T淋巴细胞亚群中转录因子BCL11B的方法。最佳方案在固定和通透步骤中均使用了皂角苷和吐温20，并且我们证明该方法对覆盖不同BCL11B表位的三种抗BCL11B抗体均有效。此外，我们还证明该方法能保留表面标志物的染色。
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