2.7. Lentiviral transduction and transient transfection of HyPer-sensor for live-cell imaging

Stable cell lines expressing HyPer in different cell organelles such as the mitochondria, the peroxisome and a membrane-tagged HyPer construct at the cytosolic face of the ER membrane were generated using lentiviral transduction [36]. For approaches with HyPer located in the cytosol, the NES (nuclear export signal) was employed and with HyPer located in the nucleus, the NLS (nucleus localization signal) was used. For all HyPer variants including the HyPer-7 constructs, 1.5 – 2 x 10⁵ cells were seeded on glass cover slips in 6-well dishes and transfected 24 h–48 h before the experiments using Lipofectamine3000 reagent (Thermo Fisher Scientific) according to the manufacturer's manual. The perifusion experiments with HyPer variants were performed according to Ref. [36] using a IX81 fluorescence microscope (Olympus, Tokyo, Japan). During HyPer-7 experiments, cells were perifused with physiological buffer solution in addition to different concentrations of H₂O₂ using a PC30 perfusion chamber (Next generation fluorescence imaging, Graz, Austria). For imaging ratiometric H₂O₂ signals within the cytosol were acquired using the highly sensitive probe: HyPer7.2-NES [37]. This probe was alternately excited at 420/40 nm and 490/15 nm with bandpass excitation filters and the emissions were collected in 3 s intervals using a 535/30 band-pass emission filter. For acquisition control the CellSens Software or Metafluor Software (Molecular Devices, San Jose, CA, USA) was used and obtained over time fluorescence intensities were analyzed (after background subtraction) by using the GraphPad Prism 5 software (San Diego, CA, USA). After recording the HyPer ratio, the curves were analyzed using a custom code in MATLAB R2017a to fit the curves and calculate the first derivative and inflection point and thus the maximal slope of each curve (Supplementary Fig. 5). The advantage of this method is that the inflection point is calculated mathematically accurate and standardized. This helps us to quantify and interpret the permeability of the different cell clones. The inflection point represents the maximum increase of the HyPer ratio over time, which allows us to draw conclusions about the transport capacity at different AQP8 expressions.