Axopatch multiclamp 700b

For each experiments, cells were electrically stimulated, as described above, either in the presence or absence of Anacardic Acid (AA, 20 nM). Lucifer yellow (2 mM in a standard intracellular solution composed by KAspartate 122 mM, KCl 20 mM, MgCl₂ 1 mM, HEPES 10 mM, EGTA 5 mM, CaCl₂ 1.6 mM, pH 7.3 KOH, was injected by a whole cell patch clamp technique (Axopatch Multiclamp 700B, Axon Instruments, USA) into clustered HL-1 cells perfused with an extracellular solution containing: NaCl 135 mM, KCl 4.5 mM, CaCl₂ 1 mM, MgCl₂ 1.8 mM, NaH₂PO₄ 0.4 mM, HEPES 10 mM, Glucose 10 mM, pH 7.4 NaOH. A patch was maintained on the target cell for 15 minutes to allow the internal pipette solution to diffuse into the patched and surrounding connected cells. Cell-to-cell transfer was monitored using an inverted microscope (NIKON TS100, Japan) equipped with fluorescent illumination and images were acquired by a digital camera (Infinity 1, Lumenera Corporation, Canada). The extent of coupled cells was determined by counting the number of adjacent cells containing the tracer and the time dependence of the spreading of the dye into the recipient cell (ImageJ, v1.28 software). Only first order recipient cells, directly in contact with the donor LY-microinjected cell, were used for kinetic evaluation.