Cell activation influences cell staining kinetics

Determination of individual cell Michaelis‐Menten constants

“…Stimulation with PHA may affect the rate constants, as already explained. However, because intracellular activity measures apparent K M , the increase in that value may be due to a decrease in the cellular viscosity after stimulation (25)(26)(27). The FDA molecule is then more mobile and the dissociation rate (k 2 ) may increase.…”

“…Cohen-Kashi et al (25) showed that the kinetics of [P] i can be described, to a good approximation, by the rate equation:…”

Prelytic Stimulation of Target and Effector Cells Following Conjugation as Measured by Intracellular Fluorescein Fluorescence Polarization

“…This seems to be insufficient in order to secure a permanent binding to the macromolecules, which is strengthened by the fact that both intracellular fluorescein and CF relatively easily leak out of the cell. 48,50 In contrast, the leakage of intracellular BCECF, which holds 4 to 5 negative surplus charges, is null and reveals FP of ϳ10% in PBS (five times greater than fluorescein) and FP of ϳ0.2 following disruption. This suggests that while at least part of the intracellular BCECF might bind to sites on macromolecules or be strongly associated to them, fluorescein and CF seem to be trapped within the cytosol.…”

“…The remaining uncaptured (free) cells were then washed away from the upper surface of the cell carrier by tangential rinsing with PBS. 50 l of 2 M FDA (Sigma, St. Louis, MO, F7378) PBS+Ca 2+ were introduced onto the trapped cells. FDA is a nonfluorescent derivative of fluorescein, which upon penetration into cells, is hydrolyzed by internal esterases and converted into fluorescent fluorescein.…”