NADH:NAD+ Ratio Imaging in Cells

The genetically encoded probes, SoNar for NADH:NAD⁺ ratio, were developed by and obtained from the Yang’s Lab (Chinese Academy of Sciences)^{11 (link)}. For A549 cybrid cells, the cells were seeded at the density of 4 × 10⁴/well in glass-bottom 24-well plates 2 days before transfection. Lipofectamine 3000 (Invitrogen #L3000001) was used for transfection according to the manufacturer’s protocol. Specifically, we used 1 µg DNA and the ratio of p3000 to the 3000 reagent was 2:1. The cells were imaged 2 days after transfection in recording media with the LSM 880 microscope using a 20x/0.8 objective lens at 37 °C. Both probes were excited at 405 and 488 separately and emitted fluorescence longer than 535 nm was collected. For fibroblasts, Human Dermal Fibroblasts Nucleofector Kit (Lonza #VPD-1001) was used for transfection according to the manufacturer’s protocol. Specifically, we used 2.5 µg of DNA for 5 × 10⁵ cells. Similarly, fibroblasts were imaged 2 days after transfection with the same conditions as A549 cybrid cells. Images were acquired using Zen Black software (Carl Zeiss) and analysed using Fiji. Regions of interest (ROI) were generated by thresholding and binarizing images and average fluorescence intensity in the ROIs for each channel were then measured. Ratios between the signal excited at 405 and 488 nm were calculated.