HKOH-1r 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	C34H25CL2I2NO11
Molecular Weight	948.2774
Exact Mass	946.889
CAS #	2138472-08-7
PubChem CID	156588681
Appearance	White to yellow solid powder
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	8
Heavy Atom Count	50
Complexity	1060
Defined Atom Stereocenter Count	0
SMILES	IC1C(=C(C([H])=C(C=1[H])OC1=C(C([H])=C2C(=C1[H])OC1=C([H])C(=C(C([H])=C1C12C2=C([H])C([H])=C([H])C([H])=C2C(=O)O1)Cl)O[H])Cl)I)O[H].O(C([H])([H])[H])C(C([H])([H])N(C(C([H])([H])[H])=O)C([H])([H])C(=O)OC([H])([H])[H])=O
InChi Key	VHPOZCLVPZPUPA-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H12Cl2I2O6.C8H13NO5/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;1-6(10)9(4-7(11)13-2)5-8(12)14-3/h1-10,31-32H;4-5H2,1-3H3
Chemical Name	2',7'-dichloro-3'-hydroxy-6'-(4-hydroxy-3,5-diiodophenoxy)spiro[2-benzofuran-3,9'-xanthene]-1-one;methyl 2-[acetyl-(2-methoxy-2-oxoethyl)amino]acetate
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: This product requires protection from light (avoid light exposure) during transportation and storage.
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. Working solution preparation for HKOH-1r 1.1: Prepare the stock solution by dissolving 1 milligram of HKOH-1r in 107 μL DMSO to create a 10 mM stock solution. Note: It is advised to keep the stock solution out of direct sunlight and between -20°C and 80°C to prevent repeated freezing and thawing. 1.2 Making the HKOH-1r working solution: Use serum-free cell culture medium to dilute the stock solution or PBS to create a working solution that is 1–10 μM. Please note: Based on the current circumstances, modify the HKOH-1r working fluid concentration. 2. Suspension cells in a 6-well plate (2.1) are stained A. After centrifuging at 1000g for three to five minutes at 4°C, discard the supernatant. Wash for five minutes each time, twice, using PBS. 1×106/mL b is the cell density. After adding 1 mL of the working solution, let it sit at room temperature for five to thirty minutes. an. After centrifuging at 400 g for three to four minutes at 4 °C, discard the supernatant. D. Wash for five minutes each time, twice, using PBS. E. Using a fluorescence microscope or flow cytometry, resuspend the cells in PBS or serum-free cell culture media. Cells that adhere 2.2 a. Adherent cells are cultured on sterile coverslips. B. Lift the coverslip out of the culture media and use the aspirator to remove any extra. an. To fully cover the cells, add 100 μL of working solution, shake gently, and then incubate for five to thirty minutes at room temperature. D. Wash for five minutes twice using a medium setting. either flow cytometry monitoring or fluorescence microscopy.

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: 95 mg/mL (101.25 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 4.75 mg/mL (5.06 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 47.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 4.75 mg/mL (5.06 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 47.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.0545 mL	5.2727 mL	10.5454 mL
	5 mM	0.2109 mL	1.0545 mL	2.1091 mL
	10 mM	0.1055 mL	0.5273 mL	1.0545 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.