Ix71 inverted fluorescence microscope system

Cells were processed for immunofluorescence staining as previously described^{26 (link)}. A tandem monomeric RFP-GFP-tagged LC3 (tfLC3) was used to monitor autophagy activity following manufacturer’s instructions. To evaluate tandem fluorescent LC3 puncta, cells were co-transfected with sh-AK2 or sh-NC after stable transfection with tfLC3, and washed with 1 × PBS at indicated time. Samples were examined and photographed under an IX71 inverted fluorescence microscope system (OLYMPUS).

The SYBR Green I/PI assay was performed to determine the live and dead cells of FL130, as previously described [35 (link)]. Briefly, SYBR Green I (10,000× stock, Invitrogen) was mixed with propidium iodide (PI, 20 mM, Sigma) into sterile distilled H₂O and vortexed thoroughly. The staining mixture was added to 50 μL FL130 cultures in 1.5 mL Eppendorf tubes, mixed thoroughly, and incubated at room temperature in the dark for 15 min. Specimens of FL130 cells were examined on an Olympus IX71 inverted fluorescence microscope system, and images were captured. Image analysis was performed using ImageJ, with green-colored cells representing live cells and red-colored cells representing total cells. The viability of FL130 cells was calculated by dividing the live cell count by the total cell count. Interaction and symbiosis between FL130 and TWY1.1 were examined with a light microscope. Images were taken from the cyanobacteria-fungi aggregates after 6 days of co-cultivation.