Fluorescence detection of CO release from 1 and 3 (50 μM) using a Nile red-based CO sensor (1-Ac) in RAW 264.7 cells. Row 1: Media control for all the experiments. Row 2: 1-Ac control after illumination for 1 h. Row 3: Compound 1 incubated for 4 h, followed by cell washes with media, introduction of 1-Ac and illumination for 1 h. Row 4: Compound 3 incubated for 4 h, followed by cell washes with media, introduction of 1-Ac and illumination for 1 h. Row 5: Compound 3 incubated for 4 h, followed by introduction of 1-Ac and illumination for 1 h (cells were not washed prior to addition of the sensor). All cells were co-stained with Hoechst 33342 nuclear dye (blue channel) to assess cell integrity and illuminated with 460 nm LED array (66,3351 lx). Green channel: Detection of fluorescence emission by 1 or 3. Red channel: Detection of CO sensor. Size of bar = 40 μm.