One day after injection of 5 ng Cx26 or Cx30 cRNA, oocytes were stripped off their vitelline membrane and paired with each other in agar wells. The next day, transjunctional conductances were measured by a dual-cell voltage clamp [21 (link)] before each ATP transfer experiment. Initial conductances were required to be between 5 and 50 μS so as to ensure a significant signal above the background while avoiding the possibility that cytoplasmic bridges could have formed between the oocytes.
Immediately following conductance measurements, one oocyte is injected with 32 nL of 1.25 mCi/mL 35S-labeled ATP-γ-S (Perkin-Elmer). Precisely 60 min after injection, the acceptor and donor cells are separated by micro-dissection under a Stereo 10X microscope. The acceptor and donor cells are immediately removed separately from the agar well and lysed with a 0.1% SDS buffer, and each is placed in 20 mL scintillation vials (Research Products International, Mount Prospect, IL, USA). A total of 10 mL of UniverSol scintillation cocktail is added (MP Biomedical), and the vials are evenly shaken prior to scintillation counting using a Beckman Coulter LS6500 scintillation counter (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Quantitative measurements of Alexa transfer through both Cx30 and 26 channels had been previously performed [22 (link)].
Immediately following conductance measurements, one oocyte is injected with 32 nL of 1.25 mCi/mL 35S-labeled ATP-γ-S (Perkin-Elmer). Precisely 60 min after injection, the acceptor and donor cells are separated by micro-dissection under a Stereo 10X microscope. The acceptor and donor cells are immediately removed separately from the agar well and lysed with a 0.1% SDS buffer, and each is placed in 20 mL scintillation vials (Research Products International, Mount Prospect, IL, USA). A total of 10 mL of UniverSol scintillation cocktail is added (MP Biomedical), and the vials are evenly shaken prior to scintillation counting using a Beckman Coulter LS6500 scintillation counter (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Quantitative measurements of Alexa transfer through both Cx30 and 26 channels had been previously performed [22 (link)].
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