Superoxide anion (O2•−). HKSOX-1m hydrobromide is a fluorescent chemosensor that specifically reacts with superoxide to generate a highly fluorescent product. It does not target a specific protein; its action is based on chemical oxidation. It is cell-permeable and can be used to monitor superoxide production in live cells. The methyl group may confer different cellular distribution (e.g., mitochondrial accumulation) compared to HKSOX-1r. The 5/6 mixture contains both regioisomers. This probe is used in flow cytometry, fluorescence microscopy, and microplate assays. There is no pharmacological target; it is a chemical reagent for detecting ROS.

At very low laser power output circumstances, HKSOX-1m(5/6-mixture) hydrobromide (10 μM) can sensitively capture the basal signals of differentiated human THP-1 cells as well as those activated by Antimycin A (1 μM). Zeiss LSM 510 Meta's mitochondrial O2 signal (with an intensity of 1%) at 514 nm excitation wavelength [1]. 1. HKSOX-1m(5/6-mixture) hydrobromide working solution preparation 1.1 Get the stock solution ready. To get a 10 mM stock solution, dilute 1 milligram of HKSOX-1m(5/6-mixture) hydrobromide with 79 μL of anhydrous DMSO. It is advised to aliquot and store the HKSOX-1m (5/6-mixture) hydrobromide storage solution at -20°C or -80°C in the dark. 1.2 Creating a functional solution Prepare a 1-10 μM HKSOX-1m (5/6-mixture) hydrobromide working solution using either heated serum-free cell culture medium or a PBS dilution stock solution. Note: Please prepare the HKSOX-1m (5/6-mixture) hydrobromide working solution for usage by adjusting its concentration to suit the current circumstances. 2. Staining suspension cells with cells 2.1 Centrifuge the cells, add PBS, and wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 Add 1 mL of the hydrobromide working solution HKSOX-1m (5/6-mixture) and let it sit at room temperature for a duration of 5 to 30 minutes. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Staining adherent cells in cells 3.1 Use sterile coverslips to cultivate adherent cells. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Cover the cells fully with 100 μL of the dye working solution, shake gently, and allow incubate for five to thirty minutes. 3.4 Use a flow cytometer or fluorescence microscope to observe after aspirating the dye working solution and washing it twice in culture media for a total of five minutes each time.

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In vitro, HKSOX-1m (5/6-mixture) hydrobromide (1-10 uM) reacts specifically with superoxide generated by xanthine/xanthine oxidase, resulting in a strong fluorescence increase (ex 510 nm, em 580 nm). The reaction is selective over other ROS (H2O2, •OH, ONOO−, NO). The presence of a methyl group may increase the rate of reaction or alter the spectral properties slightly. In cell-based assays, loading cells with HKSOX-1m (5 uM) for 30-60 min at 37degC, followed by stimulation with PMA (phorbol ester) or LPS, leads to increased fluorescence that can be detected by flow cytometry or microscopy. The hydrobromide salt enhances solubility, reducing the need for organic solvents. The probe is not cytotoxic at working concentrations. Compared to DHE, it has better specificity for superoxide. It is used to measure superoxide in immune cells, neurons, and cancer cells.

Not applicable. HKSOX-1m hydrobromide is not a drug; it is used as a probe in ex vivo and in vivo studies. It can be injected into animals to detect superoxide production in tissues. For example, in a mouse model of acute liver injury (acetaminophen overdose), i.v. injection of the probe (1-5 mg/kg) leads to fluorescence in the liver that correlates with superoxide generation. The hydrobromide salt facilitates formulation in saline. The mixture is used for research; no therapeutic application.

To validate superoxide specificity, perform a cell-free assay in a 96-well black plate. Prepare a 5 uM solution of HKSOX-1m hydrobromide in PBS (pH 7.4). Add xanthine (100 uM) and xanthine oxidase (0.1 U/mL) to generate superoxide. Incubate at 37degC for 30 min. Measure fluorescence (ex 510, em 580). A positive control with superoxide dismutase (SOD, 100 U/mL) should quench the signal. For specificity, test with H2O2 (100 uM), ONOO− (10 uM), NO (DETA-NONOate, 100 uM), and hydroxyl radical (Fe2+/H2O2). Only superoxide should give a signal. The probe is stable at 4degC in the dark. Dissolve in water or PBS from a stock in DMSO (10 mM). For hydrobromide, water solubility is high. The 5/6 mixture behaves similarly.

For cellular assays, seed RAW 264.7 cells in a 96-well black plate (2×10^4 cells/well) in DMEM with 10% FBS. Incubate overnight. Remove medium, add HKSOX-1m (5 uM) in HBSS (without phenol red). Incubate for 30 min at 37degC. Wash twice with PBS. Add PMA (100 nM) or vehicle. Incubate for 30-60 min at 37degC. Read fluorescence (ex 510, em 580) in a plate reader. For flow cytometry, use a 15 mL tube; after loading and stimulation, detach cells (trypsin), wash, and analyze on a flow cytometer (ex 488 nm, em 585 nm). For microscopy, use confocal with live cells. The hydrobromide salt is water-soluble, so DMSO concentration is low. Control groups: SOD (100 U/mL) added during stimulation to quench signal. Also use DMSO control. The probe is not cytotoxic at 5 uM for up to 4 h. All experiments in triplicate. The 5/6 mixture is used as is.

For in vivo imaging, use a mouse model of inflammation. Inject mice with LPS (10 mg/kg, i.p.) to induce systemic inflammation. After 4 h, inject HKSOX-1m hydrobromide (1 mg/kg in 100 uL sterile saline) via tail vein. After 30 min, euthanize mice, collect liver, lung, and spleen, and measure fluorescence in tissue homogenates. Alternatively, use an IVIS imager. For a local inflammation model (carrageenan paw edema), inject carrageenan into the paw, then inject the probe intravenously, and image the paw. The hydrobromide salt is suitable for injection. All animal work requires IACUC approval. The probe is for research only.

Not applicable. The probe is not a drug; pharmacokinetics are not studied. It is rapidly cleared from circulation. No ADME data.

No toxicity. At doses up to 5 mg/kg i.v. in mice, no adverse effects are reported. In cell culture, up to 50 uM for 4 h does not affect viability. Standard lab safety.

[1]. Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo. J Am Chem Soc . 2015 Jun 3;137(21):6837-43.

HKSOX-1m is a methylated variant of the HKSOX-1 superoxide probe, offering different cellular distribution (possibly mitochondrial). The hydrobromide salt increases water solubility. The 5/6 mixture is a mixture of regioisomers. It is used as a selective fluorescent indicator for superoxide in live cells. It is not a drug; for research use only.

C49H37BR2F10N2O10PS2

1258.72

HKSOX-1m (5/6-mixture);1786411-19-5

Solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~200 mg/mL (~158.89 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)