Determination of individual cell Michaelis‐Menten constants

Abstract

Background: A novel methodology for the measurement and analysis of apparent K M (Michaelis-Menten constant) and V MAX values of individual cells is suggested. It is based on a mathematical model that considers substrate influx into the cell, its intracellular enzymatic hydrolysis, and the product efflux. The mathematical formulation was approximated linearly in order to analyze intracellular substrate conversion characteristics via Michaelis-Menten theory.

Application of a Static Fluorescence‐based Cytometer (the CellScan) in Basic Cytometric Studies, Clinical Pharmacology, Oncology and Clinical Immunology

“…This supposition is well supported by both theory and experimental findings concerning changes in intracellular pH, viscosity and cell diameter upon stimulation. These findings further establish the CellScan as a tool for assessing enzymatic reaction kinetics (Sunray et al 2002).…”

Monitoring of Intracellular Enzyme Kinetic Characteristics of Peripheral Mononuclear Cells in Breast Cancer Patients

“…Measurements. The enzymatic hydrolysis of FDA and the intracellular accumulation of fluorescein by individual PBMCs were measured by the Individual Cell Scanner on a cell tray as described previously (18). The control of the cell uniformity was performed by microscopic examination on the cell tray developed and patented by our labs.…”

“…The main kinetic characteristics K m (the Michaelis constant) and V max (maximal velocity) of biochemical reaction for individual cells (maximum 300 cells/patient) were calculated after repeated periodical measurements (24 times) of the same cells, which were sequentially exposed to various increasing fluorogenic substrate concentrations (0.6 M, 1.2 M, 2.4 M, and 3.6 M) of FDA in PBS staining solutions. Cells were measured six times per each substrate concentration at time intervals of ϳ40 s. The time intervals for the replacement of FDA solutions were 50 s. Under the sequential FDA exposure, the fluorescent intensity slope between successive FDA concentrations was almost zero, which means that V 0 dropped back to zero (18). The duration of all 24 of the measurements did not exceed 1,000 s. Damaged and dead cells, which did not show an increase in fluorescent intensity on exposure to higher concentrations of FDA, were excluded from the mathematical analysis.…”

Fluctuation of Information Entropy Measures in Cell Image

“…Peripheral Blood Lymphocytes (PBL) were isolated from the heparinized blood (10 mL) of healthy subjects by the cell density Ficoll Paque gradient as previously described [32]. Following separation, cells were suspended in complete RPMI medium at 2 × 10 6 cells/mL.…”