Hydroxyphenyl Fluorescein (HPF) is a ROS fluorescent probe dye that can directly detect highly reactive oxygen species (hROS). Hydroxyphenyl fluorescein selectively and dose-dependently reacts with hROS, such as the hydroxyl radical and peroxynitrite, which exhibit strong fluorescence.

Physicochemical Properties

Molecular Formula	C26H16O6
Molecular Weight	424.402
Exact Mass	424.094
CAS #	359010-69-8
PubChem CID	16760638
Appearance	Colorless to light yellow liquid
Density	1.5±0.1 g/cm3
Boiling Point	709.9±60.0 °C at 760 mmHg
Flash Point	249.8±26.4 °C
Vapour Pressure	0.0±2.4 mmHg at 25°C
Index of Refraction	1.755
LogP	4.59
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	2
Heavy Atom Count	32
Complexity	704
Defined Atom Stereocenter Count	0
SMILES	C1=CC=C2C(=C1)C(=O)OC23C4=C(C=C(C=C4)O)OC5=C3C=CC(=C5)OC6=CC=C(C=C6)O
InChi Key	NPOSMYDPUKBGQL-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H16O6/c27-15-5-8-17(9-6-15)30-18-10-12-22-24(14-18)31-23-13-16(28)7-11-21(23)26(22)20-4-2-1-3-19(20)25(29)32-26/h1-14,27-28H
Chemical Name	3'-hydroxy-6'-(4-hydroxyphenoxy)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: 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	The HPF working solution is prepared. 1.1 Storing solution preparation Create 10 mM HPF using DMSO. It is advisable to separate the HPF storage solution and keep it in the dark at -20°C or -80°C. 1.2 Preparing the storage solution: To make a 1–10 μM HPF working solution, use either PBS diluted storage solution or preworked serum-free cell culture medium. Please be sure to utilize freshly made fluid and modify the HPF working fluid concentration based on the current circumstances. The work of cell staining 2.1 Set up the suspended cells: Centrifuge the cells to gather them, then do two 5-minute PBS washes. Replace the myocardium with islet digestive cells to create adherent cells. Add PBS and centrifuge for five minutes each time, then discard the supernatant and repeat the process twice. 2.2 Include a milliliter of HPF Utilizing Reject the supernatant after centrifuging 2.3 400 g for 3–4 minutes at 4°C. 2.4 Weigh the cells twice, giving them five minutes each time, then add PBS. 2.5 Use a fluorescent microscope or flow cytometer to detect the cells after resuspending them in 1 mL of plasma or PBS.
ln Vivo	Hydroxyphenylfluorescein (HPF) probe (15 μl, 25 μM) was inserted into the testes of anesthetized mice 20 min before IR (ionizing radiation). The buildup of ?OH was measured by the fluorescence signal generated by the oxidized form of HPF [3].
References	[1]. Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage. J Am Chem Soc. 2018 Nov 21;140(46):15957-15968.
Additional Infomation	HPF is a member of xanthenes.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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	2.3563 mL	11.7813 mL	23.5627 mL
	5 mM	0.4713 mL	2.3563 mL	4.7125 mL
	10 mM	0.2356 mL	1.1781 mL	2.3563 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.