Functional assay of the target gene transiently co-transfected with AQP4 after multiple-cycles of ultra-quick freezing/thawing.

<p>(A) Plasmid containing ET_AR-IRES-AQP4 cDNA. (B) The increased transfection efficiency in CHO cells transiently transfected with ET_AR-IRES-AQP4 by multiple cycles of ultra-quick freezing/thawing. Data are shown as the mean ± standard deviation (n ≧ 3, **P<0.01). (C) Flow-cytometry analyses quantitatively show the population of AQP4 positive cells after 3^rd ultra-quick freezing (top) or without any freezing (bottom). (D) Intracellular calcium imaging in response to endothelin-1 (ET₁). Cells were pre-loaded with the calcium indicator Oregon Green/AM. Intensity versus time traces of the cells is shown after 3^rd ultra-quick freezing (top) or without freezing (bottom). Five representative traces out of 20 measurements are shown for each group. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087644#pone.0087644.s004" target="_blank">Video S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087644#pone.0087644.s005" target="_blank">S2</a>.