Fura-2 AM fluorescence imaging was utilized to measure [[[Ca.sup.2+]].sub.i] levels as previously described [21].
Fura-2 pentakis(acetoxymethyl) (Fura-2/AM) calcium fluorescent probes were purchased from Sigma-Aldrich Co.
Twenty-four hours after dissociation, FDB fibers were incubated with 5 /M
Fura-2 acetoxymethyl ester (
Fura-2 AM; Invitrogen, Eugene, OR, USA) for 30 min at 37[degrees]C, in a buffer containing the following: 125 mM NaCl, 5 mM KCl, 1 mM MgS[O.sub.4], 1 mM K[H.sub.2]P[O.sub.4], 5.5 mM glucose, 1 mM Ca[Cl.sub.2], 20 mM HEPES, and 1% bovine serum albumin, pH 7.4.
Fura-2 fluorescence was excited alternately at 340 and 380 nm and the 340/380 ratio was obtained.
Excitation with the 360 nm filter (close to the
Fura-2 isosbestic point) allowed observation of the cells' morphology and of the changes in the concentration of the dye, irrespective of the changes in [[[Ca.sup.2+]].sub.i], while the 360/380 nm ratio allowed visualization of the [[Ca.sup.2+]].sub.i] changes in the cytoplasm.
The behavior of cytosolic calcium mobilization revealed by
fura-2 closely resembled that of elastase, thus suggesting that they may be related.
Platelet cytosolic [Ca.sup.2+] was measured with the fluorescent indicator
Fura-2 by the method described by Pollock et al.
The cells were loaded with 5.0 [micro]M
Fura-2 AM (Dojindo, Japan) in HBSS for one hour at room temperature (24 to 26[degrees]C) and washed with HBSS.
Eleven mixtures of buffer and Fura-2-AM were used to record the
Fura-2 spectrum.
Direct [Ca.sup.2+] measurement using
fura-2 dye in aortic strips revealed that arsenite enhanced vasoconstriction induced by high [K.sup.+] without concomitant increase in intracellular [Ca.sup.2+] elevation, suggesting that, rather than direct [Ca.sup.2+] elevation, [Ca.sup.2+] sensitization may be a major contributor to the enhanced vasoconstriction by arsenite.
Intracellular [Ca.sup.2+] transients were measured as changes in
fura-2 fluorescence intensity (?FFI).
This conclusion is supported by preceding studies with two calcium probes, Indo-1 and
Fura-2, which also detected no difference [11, 12].
