New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations
New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations
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DOI：
10.1002/cyto.a.20365
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发表时间：
2007-02-01
期刊：
影响因子：
3.7
通讯作者：
McCoy, Kathleen L.
中科院分区：
文献类型：
--
作者：
Creasy, Blaine M.;Hartmann, Constance B.;McCoy, Kathleen L.
背景：组织蛋白酶是内体/溶酶体蛋白酶。在调节心血管、神经、肌肉骨骼和免疫系统中的细胞生理过程中发挥重要作用的蛋白酶。病理生理过程往往与组织蛋白酶的表达和活性的变化，导致使用组织蛋白酶作为疾病标志物的诊断和预后的可能性。方法：我们描述了一种新的检测利用氩激光流式细胞仪测量半胱氨酸组织蛋白酶B，L和S在活细胞中使用细胞可渗透的荧光甲酚紫共轭肽作为选择性底物的活动。进行底物浓度依赖性和时间动力学研究。活性测定结合免疫荧光染色检测细胞系特异性分子和评估组织蛋白酶活性在一个异质性的细胞population.Results：底物浓度利用不受限制，因为MFI显着增加在巨噬细胞系刺激细菌脂多糖。选择性组织蛋白酶抑制剂证明了底物切割的选择性。分析前固定和储存的细胞没有荧光产物的损失。组织蛋白酶B，L和S的活动在脾B细胞，T细胞和巨噬细胞鉴定的免疫荧光staining.Conclusion：这种新的技术确定组织蛋白酶活性的基础上，每个细胞，而不需要纯化不同类型的细胞从一个异质性的细胞群体。(c)2007年国际分析细胞学学会。
Background: Cathepsins are endosomal/lysosomal. proteases that play important roles in regulating cell physiological processes in cardiovascular, neurological, musculoskeletal, and immunological systems. Pathophysiological processes are often associated with a change in cathepsin expression and activity, leading to the possibility of using cathepsins as disease markers for diagnosis and prognosis.Methods: We describe a new assay utilizing an argon laser flow cytometer to measure activities of cysteine cathepsins B, L, and S in live cells using cell permeable fluorogenic cresyl violet-conjugated peptides as selective substrates. Substrate concentration dependency and time kinetics studies were performed. The activity assay was combined with immunofluorescence staining to detect cell lineage-specific molecules and assess cathepsin activities in a heterogeneous cell population.Results: Substrate concentrations utilized were not limiting, because MFI significantly increased in a macrophage cell line stimulated with bacterial lipopolysaccharide. Selective cathepsin inhibitors demonstrated the selectivity of substrate cleavage. Cells fixed and stored before analysis had no loss of fluorescence product. Activities of cathepsins B, L and S in splenic B cells, T cells and macrophages identified by immunofluorescence staining were analyzed.Conclusion: This novel technique determines cathepsin activities on a per cell basis without requiring purification of different cell types from a heterogeneous cell population. (c) 2007 International Society for Analytical Cytology.