期刊
CYTOMETRY PART A
卷 71A, 期 2, 页码 114-123出版社
WILEY-LISS
DOI: 10.1002/cyto.a.20365
关键词
cysteine cathepsins; enzymatic activity-immunofluorescence; flow cytometry; proteases
资金
- NCI NIH HHS [P30CA16059] Funding Source: Medline
- NIAID NIH HHS [T32AI07407] Funding Source: Medline
- NIDA NIH HHS [P50DA05275] Funding Source: Medline
- NIEHS NIH HHS [R01ES07199] Funding Source: Medline
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据