Lungs explanted from 7–9 week human fetal tissue and E12.5 mice were cultured for 48–72 hours according to previous published protocols10 (link)27 (link)28 (link)29 (link). Human fetal lungs were separated into two halves with the trachea alternatively kept with the left or right lung in the same experimental series, a manoeuvre which did not affect lung growth. Timelapse images were captured at 0, 24, 48 and 72 hours with a dissecting microscope equipped with a digital camera (Leica Microsystems, Milton Keynes, U.K.).
For the fetal Ca2+o conditions, [Ca2+]o in the DMEM-F12 medium was increased from 1.05 mM [Ca2+]o to 1.70 mM [Ca2+]o using 1 M CaCl2 (Sigma-Aldrich, Gillingham, U.K.). Inhibitors of chloride-transporting mechanisms included the wide-spectrum anion exchange inhibitor 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (Sigma-Aldrich) and the CFTR specific inhibitor Inh-172 (Tocris) and the loop diuretic, bumetanide (Sigma-Aldrich). All chloride channel inhibitors were dissolved in DMSO, which has previously been shown not to affect lung explant growth10 (link). Vehicle control experiments were performed by adding the equivalent amount of DMSO to the lung cultures.
For the fetal Ca2+o conditions, [Ca2+]o in the DMEM-F12 medium was increased from 1.05 mM [Ca2+]o to 1.70 mM [Ca2+]o using 1 M CaCl2 (Sigma-Aldrich, Gillingham, U.K.). Inhibitors of chloride-transporting mechanisms included the wide-spectrum anion exchange inhibitor 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (Sigma-Aldrich) and the CFTR specific inhibitor Inh-172 (Tocris) and the loop diuretic, bumetanide (Sigma-Aldrich). All chloride channel inhibitors were dissolved in DMSO, which has previously been shown not to affect lung explant growth10 (link). Vehicle control experiments were performed by adding the equivalent amount of DMSO to the lung cultures.
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