HCP 

HCP is a two-photon fluorescent probe capable of crossing the blood-brain barrier, which is specifically designed for real-time detection of hypochlorous acid (HClO/ClO^-) produced by myeloperoxidase (MPO). Chlorination of the quinoline skeleton of HCP generates HCP-Cl, which eliminates intramolecular charge transfer (ICT) quenching and thereby produces fluorescence. HCP is suitable for in vivo dynamic imaging of HClO in the brains of epileptic mice and high-throughput screening of antiepileptic drugs (HCP-Cl: Ex/Em = 800 nm/495 nm)^[1].

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.

HCP (0.5 mg/kg; intravenous injection; single administration) enables highly sensitive, real-time in vivo detection of elevated hypochlorous acid (HClO) levels generated by myeloperoxidase in the brains of kainic acid (KA)-induced epileptic mice, while possessing blood-brain barrier permeability and two-photon imaging capability^[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. In Vivo Brain HClO Imaging in Mice
1.1 Animal models: KM or C57BL/6 mice; for epilepsy models, KA is administered i.p. at 6 mg/kg
1.2 Probe injection:
HCP is solubilized with normal saline containing 5-10% DMSO (or a small amount of Cremophor EL)
Administer via tail vein injection (i.v.) at a dose of 0.5 mg/kg body weight
For the epilepsy group, Apigenin (60 mg/kg i.p.) can be administered pre/post-treatment (for 3 consecutive days or after treatment)
1.3 Imaging:
Anesthetize the mice (sodium pentobarbital i.p. or Isoflurane (HY-A0134)) and fix them in position.
Time points for data collection after i.v. injection of HCP: 5, 15, 30, 45, 60 min
In vivo two-photon imaging (craniotomy/thin skull window): TPM Ex = 800 nm, collect Em at 460-520 nm (HCP-Cl green channel)
Draw ROIs in the brain (hippocampal CA1/CA3 region projection) to quantify F (KA)/F (WT).
1.4 Control: Pre-administration of 4-ABAH (MPO inhibitor) or Apigenin results in observable signal reduction.
2. Two-Photon Imaging of Mouse Brain Sections
2.1 Administer HCP (0.5 mg/kg) via i.v. injection in vivo → harvest the brain (optimal at 30-60 min post-injection).
2.2 Use a vibratome to cut 300 μm-thick coronal sections (containing the hippocampus), and continuously perfuse with ice-cold aCSF gassed with 95% O₂/5% CO₂.
2.3 Optional: Incubate the sections briefly in PBS containing HCP (10-50 μM) for 30 min (if in situ conversion is insufficient).
2.4 Wash twice with PBS.
2.5 TPM imaging: Ex = 800 nm, Em = 460-520 nm; Z-stack scanning can be performed to a depth of 150 μm.
2.6 Perform HE staining or MPO immunofluorescence co-labeling on adjacent sections for validation.

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

290.32

C₁₆H₁₄N₆

2413777-91-8

CN(C1=CC2=C(C=C1)N=C(C=C2)/C=N\C(C#N)=C(N)\C#N)C

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Shao C, et al. Epileptic brain fluorescent imaging reveals apigenin can relieve the myeloperoxidase-mediated oxidative stress and inhibit ferroptosis. Proc Natl Acad Sci U S A. 2020;117(19):10155-10164.