NIR-PN1

NIR-PN1 exhibits an ultrafast, highly selective and sensitive fluorescent response to ONOO⁻ in cell-free buffer, with a limit of detection of 4.59 nM. It remains stable over a wide pH range and shows optimal performance under physiological pH conditions^[1].
NIR-PN1 (10 μM; 30 min) penetrates PC12 cells, reacts selectively with endogenous and exogenous ONOO⁻ to generate detectable fluorescence, and exhibits low cytotoxicity against PC12 cells with an IC₅₀ of 117 μM^[1].

Guide (The following is our recommended experimental protocol. This protocol serves only as a reference guide, and specific operations should be adjusted according to your actual needs).
1. Stock Solution Preparation
Weigh out NIR-PN1 and dissolve it in anhydrous DMSO to prepare a 10 mM stock solution. Store the solution at -20℃ away from light.
Dilute the stock solution with the corresponding buffer to prepare a working solution with a concentration of 10 μM (final DMSO content ≤ 1%).
2. Cell Fluorescence Imaging (PC12 / SH-SY5Y)
2.1 Culture cells in DMEM containing 10% FBS at 37℃ in a 5% CO₂ atmosphere until the confluence reaches 60-70%.
2.2 Replace the culture medium and add NIR-PN1 to a final concentration of 10 μM (with ≤ 1% DMSO).
2.3 Incubate the cells at 37℃ for 30 min.
2.4 Wash the cells 2-3 times with PBS to remove free probes.
2.5 Perform confocal imaging with Ex = 510 nm and collect Em signals in the range of 525-700 nm (read at 670 nm).
Positive control: Pre-treat cells with SIN-1 (800 μM, 4 h) or LPS (20 μg/mL) + IFN-γ (300 ng/mL, 12 h) to induce ONOO^- production; Negative control: Pre-treat cells with uric acid UA (600 μM, 4 h) to scavenge ONOO^-.
3. Drosophila Brain Slice Staining
3.1 Dissect brains from 20-day-old wild-type / parkin-deficient Drosophila and place them in ice-cold PBS (pH 7.4).
3.2 Incubation solution: 50 μM NIR-PN1 + 1% DMSO + 0.1% Triton X-100 in PBS.
3.3 Incubate the brain slices at 25℃ for 1 h.
3.4 Wash the slices 3 times with PBS.
3.5 Perform confocal imaging using the same parameters (Ex 510 nm, Em 525-700 nm).
4. Live C. elegans Staining
4.1 Synchronize nematodes at the L4 stage (WT WLZ1 or PD model WLZ3).
4.2 Incubate the nematodes in 50 μM NIR-PN1 + 1% DMSO in water/NGM buffer for a short period (stain for 5 min as described, followed by culturing on NGM at 15℃ for 2 h).
4.3 Transfer the nematodes to a 2% agar pad and perform confocal imaging (Ex 510 nm).
5. Mouse Brain Slice Imaging
5.1 Establish MPTP-induced PD mouse models (or control mice), then sacrifice the mice by cervical dislocation and harvest their brains.
5.2 Use a vibrating microtome to cut 300 μm-thick brain slices containing the substantia nigra region, with all operations performed on ice.
5.3 Incubation solution: 50 μM NIR-PN1 + 1% DMSO + 0.1% Triton X-100 in PBS.
5.4 Incubate the slices at room temperature/37℃ for 2 h.
5.5 Wash the slices thoroughly with PBS.
5.6 Perform confocal imaging (Ex 510 nm, Em 525-700 nm), and localize the ROI to the substantia nigra region for analysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.