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 -1aurate) or umbelliferone (4-methylumbelliferyl 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.Key terms: Flow cytometry, enzyme kinetics, esterase, phosphatase, hematopoietic cells Enzymatic activities of cells or tissues can be determined by biochemical or by histochemical methods. For biochemical determination the cells or tissue are usually homogenized, the enzymatic activities can then be precisely quantified. Such measurements are, however, bulk measurements, therefore the results are difficult to interpret on a cellular level if the tissue or the cell suspension contain different cell populations. Furthermore the homogenization destroys the cells as functional units which may influence the enzymatic activities. The histochemical approach avoids these problems because enzymatic activities become morphologically apparent on a single cell level. The quantitation of histochemical stains is, however, difficult and time consuming. Since the f i s t cytochemical demonstration of esterase activity in blood cells by GomoriPresented at Automated Cytology VII, Asilomar, California, November 25-30, 1979. 1953 (5), many enzyme staining techniques mostly based on light absorption have been developed (12). The methods are applicable in investigative as well as in diagnostic pathology and play an important role in the identification of hematopoietic cells (15), the investigation and diagnosis of enzyme abnormalities (7), the recognition of cell maturation processes (11) and the classification of leukemias (2, 4). Flow cytometric measurements combine the advantages of biochemical and histologic enzyme determination. They are single cell measurements at high speed (1000-2000 cells/second) and the enzyme activity can be precisely quantified. Flow cytometers have furthermore the advantage to be capable of evaluating simultaneously other cell parameters as cell volume, DNA content or cell membrane receptors. Due to these possibilities flow cytometers can be used for automated differential white cell counting (1 The ai...

Determination of individual cell Michaelis‐Menten constants

“…The FC enables the rapid measurement of the fluores-cence intensity (FI) of a large cell population. However, because each cell in the flow is measured only once, the kinetic curves of the FC (13)(14)(15)(16)(17)(18) provide sequential measurements of single cells over a period of time but not of the same single cell. Therefore, investigating different enzyme activities in different cell types or in subcellular areas using the FC gives only an average K M value for a population of cells (18 -20).…”

Continuous measurement and analysis of staining kinetics by flow cytometry

“…Manual sampling has been used to collect cell population data at several time points during kinetic staining (11,17). Continuous quantitative description of enzyme kinetics within these cells may allow further recognition of cell subpopulations with isoenzymes of biologic or clinical significance (16).…”

“…Occasionally, multiple time points of a dynamic dye-uptake process are sampled by manual collection of FCM data from time-defined subpopulations (6,11,17). Martin and Swartzendruber (13) described a time-base generator which allows time to be used as a true parameter in FCM multi-parameter analysis and so allows FCM analysis of the dynamics of cell staining.…”

Realtime acquisition, storage, and display of correlated three‐parameter flow cytometric data

“…The data projections may also be displayed: as l-paraThe acquisition of correlated three-parameter data from flow cytometers has virtually always required the use of "list-mode" techniques. These list-mode data would be subsequently retrieved, grouped, and analyzed (off-line) by large minicomputers or mainframe computers (15,20,22) for the construction of three-parameter contour maps (15,221, three-parameter monochrome scattergrams (201, or three two-parameter projections of the data (1,3,4,15). In contrast, a modular, nonprograrnmable device for on-line three-parameter collection and monochrome display has been recently reported (lo), but no data were shown.…”

“…"List-mode" techniques (9,15,20,22) are not used for any mode of data-collection or display generation. No "gating" of any parameter by another is necessary, although this can be done retrospectively by the computer.…”