GCaMP 3 (gift from Michael Laflamme Lab, Toronto)44 (link),47 (link) and H9, CF01 iPSC were differentiated to cholangiocyte cysts and plated down in Lab-Tek Chamber Slide (Millipore) or in flow chamber (ibidi) 2 days prior to the assay. Day 27 hepatoblasts were aggregated and plated down 2 days prior to the assay. Chambers were pre-coated with fibronectin. The cells were replaced in Tyrode buffer before the assay. H9-derived-cholangiocytes and CF01 derived cholangiocytes were stained with 10 μM Fluo4 (Invitrogen) with 0.04% Pluronic F127 (Invitrogen) for 30 min prior to the assay. To measure calcium response to ATP and TUDCA (Sigma) in 3D cysts, GCaMP derived cholangiocyte 3D cysts were embedded in the type1 collagen gel just before the assay.
Calcium response to 20 μM ATP (Sigma) or flow (Perista BioMini Pump: ATTO) were analyzed using a confocal fluorescence microscope (NIKON A1 Resonant Confocal Microscope) and images captured using the Nikon Elements software.
Calcium response to 20 μM ATP (Sigma) or flow (Perista BioMini Pump: ATTO) were analyzed using a confocal fluorescence microscope (NIKON A1 Resonant Confocal Microscope) and images captured using the Nikon Elements software.
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