Seradish peroxidase-conjugated secondary antibody from Amersham Biosciences (Buckinghamshire, UK) was utilized to detect all bound main antibodies. Reporter gene assay. The promoter area with the rat early growth response gene-1 (egr-1) gene ( 525 to 117) (Changelian et al., 1989) was obtained by PCR and subcloned into pGL3-Basic (Promega, Madison, WI). This reporter gene vector was transfected, employing TransFast (Promega), into astrocytes that had been grown for 48 hr in DMEM containing 25 mM HEPES, pH 7.four, and 1 FCS. Following 24 hr, the medium was changed to GF-free ADM, then, after 48 hr culture, with or without the need of pretreatment, as described for the Western blot experiments above, GFs have been added for 6 hr, and luciferase activity was assayed utilizing PicaGene (Nippon Gene, Tokyo, Japan). Slice culture and calcium imaging. Slice cultures were prepared from the hippocampus of postnatal day 7 Wistar rats, as described previously (Hirasawa et al., 2000), and cultured for 74 d prior to calcium imaging. BSS containing 0.1 mM ascorbic acid and 0.five mM inositol was utilized all through, and sulfinpyrazone was incorporated as described for the cell culture experiments. The cells have been incubated with 50 M MK801 for 30 min ahead of and during loading for 1 hr at 37 with fluo-4 AM (Molecular Probes, Eugene, OR) in BSS containing 0.005 Cremophore. Following 3 washes, the slices have been incubated for 30 min at area temperature in medium without MK801 and then had been transferred for five min to BSS containing 100 mM mannitol, which suppresses swelling for the duration of pharmacological stimulation. Calcium Enolase medchemexpress imaging was performed applying an E600FN upright CYP26 manufacturer microscope in addition to a Fluor 40 /0.8w objective (each from Nikon, Tokyo, Japan) equipped with a CSU-10 laser confocal scanning unit (Yokokawa, Tokyo, Japan), a 532R-BS-A04 argon laser (Melles Griot, Irvine, CA), as well as a C6790 CCD camera (Hamamatsu). Fluorescence pictures had been acquired employing AQUACOSMOS software (Hamamatsu), and the fluorescence ratio (F/Fo) was calculated from the typical intensity of the indicated areas.Growth factor-induced calcium oscillation As inside a earlier report (Jensen and Chiu, 1990), astrocytes cultured in medium containing 10 FCS, a normally used additive, have been identified to consist of a mixture of two populations, the proportions of which varied in between cultures. Certainly one of these showed a transient response, along with the other an oscillatory response, to glutamate (30 M) or ATP (100 M) (Fig. 1 A, best panels); the percentage of responding cells displaying oscillatory responses to glutamate or ATP, respectively, was 33.three (n 42) and 18.9 (n 58). In contrast, following culture for 48 six hr in serum-free defined medium containing EGF and bFGF (ADM), nearly all of the responding cells showed calcium oscillationResults10946 J. Neurosci., November 26, 2003 23(34):10944 Morita et al. Dual Regulation of Astrocytic Calcium Oscillation(center panels). Common imaging information for the calcium oscillation in response to glutamate are shown inside the supplementary data (movie 1; obtainable at www.jneurosci.org). Furthermore, these cells showed a comparable oscillatory response to thimerosal (ten M), which affects the redox state in the inositol-1,4,five triphosphate (IP3) receptor and induces calcium release (Swann, 1991). In contrast, cells in GF-free ADM gave a transient response to all 3 stimuli (bottom panels). The percentage of responding cells showing oscillatory responses to glutamate, ATP, or thimerosal, respectively, was ten.3 (n 156), eight.three (n 60), and 3.6 (.