PMC:4385177 / 28084-30178 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4385177","sourcedb":"PMC","sourceid":"4385177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4385177","text":"Summary of the Functional Studies in Hpca-Knockdown Neuronal-Astrocytic Co-cultures\nAstrocytes and neurons from primary cortical co-culture were loaded for 30 min at room temperature with 5 μM fura-2 AM and 0.005% pluronic acid in a HEPES-buffered salt solution composed of 156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES (pH was adjusted to 7.35 with NaOH). Fluorescence measurements were obtained on an epifluorescence inverted microscope equipped with a 20× fluorite objective. [Ca2+]c was monitored in single cells with excitation light provided by a Xenon arc lamp, and the beam passed through a monochromator at 340 and 380 nm (Cairn Research). Emitted fluorescence light was reflected through a 515-nm longpass filter to a charge-coupled-device camera (Retiga, QImaging) and digitized to a 12-bit resolution. All imaging data were collected and analyzed with software from Andor IQ. The fura-2 data were not calibrated in terms of [Ca2+]c because of the uncertainty arising from the use of different calibration techniques. Areas for the analysis were chosen depending on the GFP fluorescence intensity, and four independent experiments were performed for each condition. The figure shows representative traces of [Ca2+]c response to physiological stimuli as measured by changes in fura-2 fluorescence intensity. Compared to neurons from the scrambled or empty control (A, black triangle trace), Hpca-knockdown neurons showed no rise in [Ca2+]c in response to depolarization of the plasma membrane with 50 mM KCl (A, dark-gray trace; B, dark-gray bars). In addition, the amplitude of the response to physiological concentration of glutamate (5 μM) was lower in the Hpca-knockdown neurons than in control cells (B, black bars), although this decrease was not statistically significant. Hpca knockdown also diminished the amplitude of the [Ca2+]c response of astrocytes to an ATP stimulus (100 μM) (C and D, light-gray trace and bars). Error bars represent the SEM, and asterisks represents statistical significance (∗∗∗p \u003c 0.0001, ∗p \u003c 0.05).","divisions":[{"label":"p","span":{"begin":0,"end":83}}],"tracks":[]}