Ibitreat microscopy chamber

Image acquisition of living cells stably transfected with roGFP2 or Grx1-roGFP2 constructs were performed in μ-Slide eight well or μ-Slide VI channel, ibiTreat microscopy chambers (Ibidi, Munich, Germany) and maintained for 48 h. The cells were washed twice with Dulbecco's phosphate-buffered saline supplemented with 5% FBS and 10 mmol/L glucose prior to imaging. Perfusion of cells cultured in channels was performed with a PHD Ultra syringe pump (Harvard Apparatus, Holliston, MA, USA) at 200 μL/min. Images were collected as described previously.^{38 (link)}To visualize spatiotemporal changes of glutathione poise, raw data were exported to Zen 2009 software (Carl Zeiss) and converted into 395 nm and 494 nm sets of 12-bit TIF images. Next, the pixels belonging only to the probe were selectively segmented.⁴⁰ The calculated threshold was scaled by a fixed number for all images and the function “graythresh” was implemented in a home-written script in MATLAB (Natick, MA, USA). The pixel-by-pixel 395/494 nm false-color ratio pictures were directly calculated by dividing 395 nm on 494 nm images.
To confirm mitochondrial localization of the mtGrx1-roGFP2 probe in stably transfected CHO cells, 3D images were acquired on a Zeiss ELYRA super-resolution microscope.