B filter to ly

The functional state of gap junctions was evaluated as previously described (Martínez and Sáez, 1999 (link)) in neurosphere-derived cells and in co-cultures of neurosphere-derived cells with astrocytes or microglia. Single NPCs were iontophoretically microinjected with a glass micropipette filled with 75 mM LY (5% w/v in 150 mM LiCl). Dye coupling index was calculated as the mean number of cells to which the dye spread occurred in 3 min. All microinjections were performed in ${\text{HCO}}_3^{-}$ free F-12 medium buffered with 10 mM HEPES (pH 7.4) containing 200 μM La³⁺ to avoid cell leakage of the microinjected dye via hemichannels. Dye coupling was assessed in the absence and in the presence of 750 μM octanol. Fluorescent cells were observed using a Nikon inverted microscope equipped with epifluorescence illumination (Xenon arc lamp) and Nikon B filter to LY (excitation wavelength 450–490 nm; emission wavelength above 520 nm) and XF34 filter to DiI fluorescence (Omega Optical, Inc., Brattleboro, VT, USA). Photomicrographs were obtained using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Five to ten experiments were performed for every type of culture and dye coupling was tested by microinjecting a minimum of 10 cells per experiment.

The functional state of GJCs was evaluated as described (Figueroa et al., 2013 (link)). Briefly, confluent HeLa cell cultures grown on coverslips were used in each experiment. Single cells were iontophoretically microinjected with a glass micropipette filled with 75 mM Etd in water or Lucifer yellow (LY, 5% w/v in 150 mM LiCl). Dye coupling index was calculated as the mean number of cells to which the dye spread occurred. All microinjections were performed in ${\mathrm{H}\mathrm{C}\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ -free F-12 medium buffered with 10 mM HEPES (pH 7.4) containing 200 μM La³⁺ to avoid cell leakage of the microinjected dye via HCs. Fluorescent cells were observed using a Nikon inverted microscope equipped with epifluorescence illumination (Xenon arc lamp) and Nikon B filter to LY (excitation wavelength 450–490 nm; emission wavelength above 520 nm) and XF34 filter to Etd fluorescence (Omega Optical, Inc., Brattleboro, VT, USA). Photomicrographs were obtained using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). In all experiments, dye coupling was tested by injecting a minimum of 14 cells.

Cells plated on glass coverslips were bathed with recording medium ( ${\text{HCO}}_{\text{3}}^{-}$ free F-12 medium buffered with 10 mM HEPES, pH 7.2) and permeability mediated by gap junctions was tested by evaluating the transfer of LY to neighboring cells. Briefly, single ECs were iontophoretically microinjected with a glass micropipette filled with 75 mM LY (5% w/v in 150 mM LiCl) in recording medium containing 200 µM La³⁺ to avoid cell leakage of the microinjected dye via hemichannels, leading to underscore the extent of dye coupling. Fluorescent cells were observed using a Nikon inverted microscope equipped with epifluorescence illumination (Xenon arc lamp) and Nikon B filter to LY (excitation wavelength 450–490 nm; emission wavelength above 520 nm) and XF34 filter to DiI fluorescence (Omega Optical, Inc., Brattleboro, VT, USA). Photomicrographs were obtained using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Three minutes after dye injection, cells were observed to determine whether dye transfer occurred. The incidence of dye coupling was scored as the percentage of injections that resulted in dye transfer from the injected cell to more than one neighboring cell. Three experiments were performed for every treatment and dye coupling was tested by microinjecting a minimum of 10 cells per experiment.