Flow cytometric determination of esterase and phosphatase activities and kinetics in hematopoietic cells with fluorogenic substrates.

Abstract

Viable and formaldehyde fixed rat bone marrow or spleen cells and rat or human blood leukocytes were incubated with fluorescein (fluorescein-diacetate, -dibutyrate and -laurate) or umbelliferone (4-methyl umbelliferyl acetate and -phosphate) fluorogenic substrates for subsequent flow cytometric determination of cell fluorescence and cell volume. Formaldehyde-fixed cells preserved between 14 and 20% of the enzyme activity of the unfixed cells and the number of cell clusters for fixed and unfixed cells was the same. The esterase substrates revealed one cell cluster for spleen cells, two for bone marrow cells and four for peripheral blood leukocytes. Phosphatase activity was only associated with a cell cluster of large cells. The time course of substrate cleavage was linear during the first 10 min of incubation. Later on a plateau was reached. Specific enzyme activities were calculated on a single cell level from the simultaneous cell volume and cell fluorescence measurement. Two enzyme activities could be measured simultaneously by using a substrate mixture of umbelliferone acetate and fluorescein acetate.