New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations.
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.


CONCLUSIONS
This novel technique determines cathepsin activities on a per cell basis without requiring purification of different cell types from a heterogeneous cell population.
Creasy
Blaine M
BM
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.
Hartmann
Constance B
CB
White
Frances K Higgins
FK
McCoy
Kathleen L
KL
D000818:Animals; D002403:Cathepsins / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D013154:Spleen / Q000166:cytology / Q000201:enzymology; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D002403:Cathepsins