Axiocam mrm monochrome digital camera r3

GJ permeability was evaluated at room temperature (RT) using the scrape loading/dye transfer (SL/DT) technique [93 (link),94 (link)]. Briefly, MCs cultures were washed for 10 min in HEPES-buffered salt solution containing the following (in mM): 140 NaCl, 5.5 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 glucose, 10 HEPES, pH 7.4, followed by washing in a Ca²⁺-free HEPES solution for 1 min. Then, a razor blade cut was made in the monolayer in a HEPES-buffered salt solution with normal Ca²⁺ concentration containing the fluorescent dye Lucifer yellow (LY). After 1 min, LY (100 μM) was washed out several times with HEPES buffered salt solution. At 8 min after scraping, fluorescent images were captured using a Zeiss Axio Observer D.1 Inverted Microscope with a Solid-State Colibri 7 LED illuminator and with a 10× objective. Changes were monitored using an AxioCam MRm monochrome digital camera R3.0 (Carl Zeiss AG, Zeiss, Oberkochen, Germany), and Software ZEN Pro (Zen 2.3 [blue edition], Carl Zeiss AG, Oberkochen, Germany) for image acquisition and analysis. For each trial, data were quantified by measuring fluorescence areas in three representative fields. Quantification of changes in GJ communication induced by different treatments was performed by measuring the fluorescence area, expressed as AU [93 (link),94 (link)].

Gap junction permeability was evaluated at room temperature using the scrape-loading/dye transfer (SL/DT) technique. Briefly, cells cultures were washed for 10 min in HEPES-buffered salt solution containing the following (in mM): 140 NaCl, 5.5 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 glucose, 10 HEPES, pH 7.4 followed by washing in a Ca²⁺-free HEPES solution for 1 min. Then, a razor blade cut was made in the monolayer in a HEPES-buffered salt solution with normal Ca²⁺ concentration containing 10 µM DAPI. After 3 min, DAPI was washed out several times with HEPES-buffered salt solution. At 15 min after scraping, fluorescent images were captured using a a Zeiss Axio Observer D.1 Inverted Microscope with a Solid-State Colibri 7 LED illuminator and with a 20x objective. Changes were monitored using an AxioCam MRm monochrome digital camera R3.0 (Carl Zeiss AG, Zeiss, Oberkochen, Germany), and Software ZEN Pro (Zen 2.3 [blue edition], Carl Zeiss AG, Oberkochen, Germany) for image acquisition and analysis. For each trial, data were quantified by measuring fluorescence areas in three representative fields. Quantification of changes in gap junctional communication induced by different treatments was performed by measuring the fluorescence area, expressed as arbitrary units (AU).