1：Experimental Design and Method Selection:

Rational design of four BODIPY-based fluorescent probes (BDP-NO, Mito-NO-T, Mito-NO, Lyso-NO) using N-nitrosation strategy for NO detection. Synthesis involved nucleophilic substitution, click chemistry, reductive amination, and alkylation. Spectroscopic evaluation (UV-vis, fluorescence) in PBS-CH3CN solution. Cell imaging using confocal microscopy.

2：Sample Selection and Data Sources:

HeLa cells (human cervical cancer cell) and RAW

3：7 macrophages cultured in DMEM medium with 10% FBS. Chemical analytes (H2O2, ClO-, 1O2, O2?-, OH·, ONOO-, MGO, AA, DHA, Cys, Hcy, GSH, NO2-, Br-, I-, HCO3-, Cl-, DEA·NONOate) prepared in PBS buffer. List of Experimental Equipment and Materials:

2 Varian Model Mercury 400 MHz or 600 MHz spectrometer (1H-NMR), Varian Model Mercury 150 MHz spectrometer (13C-NMR), Agilent 6120 Quadrupole LC-MS spectrometer, AB SCIEX TRIPLE TOF 5600+ mass spectrometer (ESI-HRMS), 759S UV-visible spectrophotometer (Lengguang Tech), F98 fluorescence spectrophotometer (Lengguang Tech), Millipore elix / RIOs water purified system, confocal laser scanning microscope (Carl Zeiss LSM 710), CytoFLEX S cytometer (Beckman), Mito-Tracker Deep Red FM, Lyso-Tracker Red, Annexin V-FITC and PI (Beyotime).

4：Experimental Procedures and Operational Workflow:

Probe synthesis and purification. Spectroscopic measurements: probes (10 μM) in PBS-CH3CN (pH

5：4) with analytes, fluorescence at 515 nm (ex 495 nm). Cell viability (MTT assay), apoptosis (Annexin V/PI staining), cell imaging:

incubation with probes (10 μM) and DEA·NONOate (200 μM), co-staining with organelle trackers.

6：Data Analysis Methods:

Fluorescence intensity analysis, detection limit calculation (3σ/k), overlap coefficient for co-localization, statistical analysis from replicate experiments.