HKOH-1 is a highly sensitive green fluorescent probe that specifically detects ·OH in living cells. The maximum excitation wavelength and emission wavelength are 500 nm and 520 nm respectively.

Physicochemical Properties

Molecular Formula	C26H12CL2I2O6
Molecular Weight	745.084830284119
Exact Mass	743.81
CAS #	2031170-96-2
PubChem CID	122647002
Appearance	White to off-white solid powder
LogP	7.5
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	2
Heavy Atom Count	36
Complexity	841
Defined Atom Stereocenter Count	0
SMILES	C12(C3=C(C=C(OC4=CC(I)=C(O)C(I)=C4)C(Cl)=C3)OC3=C1C=C(Cl)C(O)=C3)C1=C(C=CC=C1)C(=O)O2
InChi Key	BZWBSIJSVLAKAF-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H12Cl2I2O6/c27-16-7-14-21(9-20(16)31)35-22-10-23(34-11-5-18(29)24(32)19(30)6-11)17(28)8-15(22)26(14)13-4-2-1-3-12(13)25(33)36-26/h1-10,31-32H
Chemical Name	2',7'-dichloro-3'-hydroxy-6'-(4-hydroxy-3,5-diiodophenoxy)spiro[2-benzofuran-3,9'-xanthene]-1-one
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

1. Preparation of HKOH-1 working solution 1.1 Get the stock solution ready. To get a 10 mM stock solution, dilute 1 milligram of HKOH-1 with 135 μL of anhydrous DMSO. It is advised to aliquot and store HKOH-1 storage solution at -20°C or -80°C in the dark. 1.2 Creating a functional solution Use preheated serum-free cell culture medium or PBS dilution stock solution to prepare a 1-10 μM HKOH-1 working solution. Note: Please prepare the HKOH-1 working fluid for usage by adjusting the concentration to suit the current circumstances. 2. Cell staining (suspension 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 HKOH-1 working solution, then let it sit at room temperature for five to thirty 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.

References

[1]. HKOH-1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells. Angew Chem Int Ed Engl. 2017 Oct 9;56(42):12873-12877. [2]. HKOH-1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells. Angew Chem Int Ed Engl. 2017 Oct 9;56(42):12873-12877.

Solubility Data

Solubility (In Vitro)

DMSO: 180 mg/mL (241.58 mM)

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3421 mL	6.7107 mL	13.4214 mL
	5 mM	0.2684 mL	1.3421 mL	2.6843 mL
	10 mM	0.1342 mL	0.6711 mL	1.3421 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.