Plasma AM concentration was measured using an immunoenzymometric assay kit [ 14].
Optimal dye incubation conditions were empirically determined to be a final FluoZin-3, AM concentration of 2 µM at room temperature for 30 min. Cells were then washed twice with PBS and then incubated for 30 min with PBS to allow complete de-esterification of intracellular AM esters.
Calculations of AM concentration utilized the standard curve included in the kit and were conducted according to the manufacturer's protocol.
AM concentration in conditioned media was measured using a human AM (1-52) enzyme immunoassay kit from Phoenix Peptides (Karlsruhe, Germany) according to manufacturer's instructions.
Finally, for the rest of the cell population (15.8%, 12 out of 76 cells in 3 slices), the frequency was a decreasing function of fluo-4 AM concentration (Fig. 5E and F; F5,33 = 7.74, P<.05), while the amplitude could be decreasing, unchanging, or increasing, depending on the individual cells.
For the majority of the cells (48.7%, 37 out of 76 cells in 3 slices), the amplitude as well as the frequency of Ca2+ spikes were an increasing function of fluo-4 AM concentration (Fig. 5A and B; Frequency: F5,105 = 14.6, Amplitude: F5,105 = 3.8, P<.05).
