Neurobiotin dye

Astrocyte monocultures were grown on 13 mm coverslips (VWR) for 5–7 days; on the day of injection, the cells were pre‐treated with 20 µM TAT‐GAP19 (Tocris) for 10 min prior to, and during microinjection to reduce dye uptake through Cx43‐containing hemichannels. A single cell was then microinjected with 0.5% lucifer yellow dye (Invitrogen) and 2% neurobiotin dye (Vector) in PBS using the FemtoJet microinjector system (Eppendorf) combined with FemtoTips II (Eppendorf). Injection pressure was set to 100 hPa for 0.1 s injection, constant pressure was kept at 10 hPa, and the needle was left in the injected cell for 5 min at room temperature. Cells on coverslip were then fixed in 4% PFA for 10 min and stained for further microscopic analysis. Images were analyzed in ImageJ software; injection areas were automatically thresholded and overlayed on the DAPI channel, after which the number of nuclei within the injection area were counted.
List of antibodies/dyes for microinjected area staining (all used at 1:500 final dilution):

Anti‐lucifer yellow rabbit – Invitrogen A‐5750

Cy3‐Streptavidin – Vector SA‐1300‐1

The cells were cultured with normal culture medium on glass coverslips until 80%–90% confluence was reached. Then, the glass coverslips were transferred to a bath solution containing 140 mM NaCl, 1 mM MgCl₂, 5.4 mM KCl, 1.2 mM CaCl₂, and 10 mM HEPES (pH 7.2). The cells were impaled with a micropipette filled with a patch pipette solution and 4% neurobiotin dye (Mr = 322.8, charge  = +1; Vector Laboratories, Burlingame, CA, USA) or 5% lucifer yellow dye (Mr = 457, charge  = −2; Invitrogen). The patch pipette solution contained 140 mM KCl, 1 mM MgCl₂, 5 mM NaCl, and 10 mMHEPES (pH 7.4). The dye solution was microinjected for 3 min with a Picospritzer (model PLI-188; Nikon, Tokyo, Japan).
After neurobiotin dye injection, the cells were fixed, permeabilized, and stained with Cy3-streptavidin conjugate (Sigma) for tracer detection. However, after lucifer yellow dye injection, the cells required only fixation. Both types of cells were then photographed with fluorescence microscopy. The adjacent cells stained by the dye were counted to determine the extent of the intercellular tracer transfer. Three independent stable-cell clones were all used to perform the dye transfer assay. The number of coupled cells was expressed as the means ±SDs. Statistical analysis was performed with Student's t-test.