Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry.
This study was designed to explore the utility of a microscope-based cytofluorometer, the laser scanning cytometer (LSC), for time-resolved kinetic measurements. This instrument measures fluorescence of individual cells rapidly, with high sensitivity and accuracy. Also recorded in a list mode fashion are the spatial X-Y coordinates of the cell on the slide as well as the time of individual cell measurement. Repeated measurement of each of a group of cells within a selected area of the slide, thus, yields information on their fluorescence parameters (integrated value, maximal pixel intensity, or fluorescence area) as a function of time. Using the fluorogenic substrate di-(leucyl)-rhodamine 110, we measured the kinetic activity of L-aminopeptidase in HL-60 cells and in monocytes, granulocytes, and lymphocytes from human blood. Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was measured in HL-60 cells. Also studied was the rate of uptake of the lysosomo-trophic fluorochrome acridine orange (AO) by human leukocytes. Several hundred cells per sample were measured rapidly with a time resolution of 20-60 s. The resolution was inversely proportional to the number of cells within the measured population. The kinetic curves constructed for individual cells had clearly defined slope and plateau regions. During data analysis the kinetic plots were matched with the respective cells; the latter were identified by their position on the slide and classified by their fluorescence or image after staining with Giemsa. Great intercellular variability was noted in enzyme kinetics among cells of the same type, and differences were seen in rate of AO uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our results indicate that LSC can be used to rapidly measure the rate of uptake or accumulation of a particular fluorochrome or the kinetics of enzymatic reactions in individual cells of large populations to reveal intercellular variability or the presence of a cell subpopulation with different kinetic properties.
Bedner
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Brander Cancer Research Institute, Department of Pathology, New York Medical College, Valhalla 10523, USA.
Melamed
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MR
Darzynkiewicz
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Z
D000165:Acridine Orange / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007834:Lasers; D012235:Rhodamines / Q000378:metabolism
D004798:Enzymes; D005452:Fluoresceins; D012235:Rhodamines; D004950:Esterases; D000165:Acridine Orange; C018506:diacetylfluorescein