title: Dynamic assay of enzyme activities in single cells by flow cytometry.
abstract: Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D006224:Cricetinae; D003584:Cytological Techniques; D004950:Esterases / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008113:Liver Neoplasms; D010053:Ovary; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D007770:L-Lactate Dehydrogenase; D004950:Esterases; D000135:Acid Phosphatase