Zen blue microscope imaging software

To examine LCN2 and EGFR colocalization, OSCC cells were plated on glass-bottomed dishes (#D35-20–0-TOP, Cellvis). The cells were then transfected with the indicated plasmids by using Lipofectamine 3000 (Invitrogen). After transfection for 24 h, the samples were observed and captured on a Zeiss microscope.
For time-lapse imaging of NP uptake by OSCC cells, GFP-endo14 labeled CAL-27 cells at approximately 30–40% confluency were seeded on glass-bottomed dishes for 16 h. After the medium was replaced with 2 mL of fresh medium, glutathione-responsive NPs (G-NPs) loaded with Cy5-labeled siLCN2 were added at an siRNA concentration of 50 nM, and the cells were incubated for 8 h. Images were captured for at least 8 h.
The captured images were analyzed by the Zen Blue microscope imaging software (Zeiss) or ImageJ program. Dozens of cells were selected to define regions of interest (ROIs) for analysis.

For detection of γH2AX, SERPINB3 and USP1 in HNSCC cells, 1 × 10⁴ cells were plated on glass‐bottomed dishes (#D35‐20‐0‐TOP, Cellvis). The cells were then treated with cisplatin (5 µm mL⁻¹) for 24 h. Then, the medium was removed and replaced with culture medium, and the cells were incubated for 24 h before collection.
After fixation with 4% paraformaldehyde and permeabilization with 0.3% Triton X‐100, the cells were blocked in 0.5% BSA–PBS for 1 h. Primary antibodies (γH2AX, SERPINB3, USP1, 1:100) were used for incubation with the cells at 4 °C overnight. The next day, after washing with PBST, the cells were stained with secondary antibodies (1:500) and DAPI (1:1000). Fluorescence images were taken on a confocal laser microscope (Zeiss LSM 800).
For time‐lapse imaging (NP uptake) of HNSCC cells, GFP‐endo14‐labeled CAL‐27 cells at ≈30–40% confluency were seeded on glass‐bottomed dishes for 16 h. After the medium was replaced with 2 mL of fresh medium, NPs loaded with Cy5‐labeled siSERPINB3 were added at a siRNA concentration of 50 nm, and the cells were allowed to incubate for 8 h during confocal capture.
The captured images were analyzed with the Zen Blue microscope imaging software (Zeiss) or ImageJ program.