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. Further protocols information, click here.