HCP

HCP (10-50 μM) is a two-photon fluorescent probe with high sensitivity, high selectivity and good stability. After reacting with HClO under cell-free in vitro conditions, it undergoes a rapid blue shift and exhibits a 138-fold fluorescence enhancement. The limit of detection after reaction with HClO is 104 nM, and the maximum two-photon absorption cross-section at a wavelength of 800 nm is 13.4 GM^[1].
HCP (0-150 μM; 48 h) exhibits only extremely low cytotoxicity against human neuroblastoma SH-SY5Y cells (at a maximum concentration of 150 μM for 48 h)^[1].
HCP (10 μM; 30 min) effectively penetrates the cell membrane of human neuroblastoma SH-SY5Y cells, detects exogenous HClO in a concentration-dependent manner, and enables specific visual imaging of endogenous HClO generated by MPO activity in living cells^[1].
HCP (10 μM; 30 min), combined with high-content analysis technology, serves as an effective tool for high-throughput screening of reagents that regulate endogenous HClO levels in human neuroblastoma SH-SY5Y cells^[1].
HCP (10 μM; 30 min) specifically visualizes endogenous hypochlorous acid (HClO) produced by MPO in human neuroblastoma SH-SY5Y cells with oxidative stress induced by glutamate or kainic acid (KA)^[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 HCP and dissolve it in anhydrous DMSO to prepare a 5-10 mM stock solution. Store the solution at -20℃ away from light.
Dilute the stock solution with PBS or complete medium to prepare a working solution with a concentration of 10 μM (final DMSO content ≤ 0.5-1%).
2. Cell Fluorescence Imaging (Neuronal Cells such as SH-SY5Y)
2.1 Seed cells in confocal dishes or 24-well plates (with coverslips), and culture them at 37℃ in a 5% CO₂ atmosphere until they reach 60-80% confluency.
2.2 For the HClO induction group: pretreat cells with LPS (HY-D1056) (1 μg/mL, 12 h), kainate (KA), or exogenously added NaOCl (HClO donor, final concentration 0-50 μM).
For the MPO inhibition control: pre-incubate cells with 4-ABAH (100 μM) or SHA.
2.3 Aspirate the old medium and add fresh medium containing HCP at a final concentration of 10 μM (with ≤1% DMSO).
2.4 Incubate the cells at 37℃ for 30 min.
2.5 Wash the cells 2-3 times with PBS.
2.6 Perform confocal imaging: use an excitation wavelength (Ex) of 405 nm (or 488 nm), collect fluorescence emission (Em) at 500-550 nm, and measure the intensity at 495 nm.
2.7 Quantification: Draw regions of interest (ROI) using ImageJ to analyze the average fluorescence intensity.
3. High Content Analysis (HCA) / High Throughput Screening (HTS) of Natural Products
3.1 Seed SH-SY5Y cells in 96-well black-walled clear-bottom plates (5000-8000 cells per well) and culture them for 24 h.
3.2 Add the natural product to be tested (e.g., Apigenin (HY-N1201) at 20 μM) and pre-incubate the cells for 12 h.
3.3 Stimulate the cells with LPS (1 μg/mL) or KA to induce endogenous MPO/HClO production (no stimulation for the control group).
3.4 Replace the medium, add HCP at 10 μM, and incubate for 30 min.
3.5 Wash the cells twice with PBS.
3.6 Use an HCA system (e.g., Operetta/ImageXpress) to collect images with an excitation wavelength (Ex) of 405 nm and an emission (Em) channel of 490-520 nm.
3.7 Quantify the average fluorescence intensity of each well, and calculate the HClO inhibition rate using the formula: HClO inhibition rate = (F_control − F_treated)/F_control × 100%.

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