Fluorescence imaging of cytosolic [Ca2+]

Cells were incubated in standard external medium (SEM) containing 145 mM NaCl, KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM Hepes 10, pH 7.42), and later loaded with the fura2/AM dye (4 μM) for 60 min at room temperature. Then, coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same standard medium pre-warmed at 37 °C and epi-illuminated alternately at 340 and 380 nm. Light emitted at 520 nm was recorded every 5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Perfusion was stopped before adding vehicle or LPS during times indicated in the figures. Pixel by pixel ratios of consecutive frames were captured, and [Ca²⁺]_cyt of regions of interest corresponding to individual neurons selected by morphometric characteristics were expressed as the ratio of emission following excitation at 340 and 380 nm (Ratio F340/F380), as reported in detail previously [28]. Analysis of responses in non-neuronal cells was also carried out by selecting cells not showing the morphology of neurons. In addition, the glial phenotype was confirmed by the lack of Ca²⁺ responses to N-methyl d-aspartate (NMDA).