Hydroxyphenyl Fluorescein (solution) 

Hydroxyphenyl Fluorescein (HPF) (solution) is a stable ROS fluorescent probe dye. Hydroxyphenyl Fluorescein has stronger specificity and stability than H2DCFDA (HY-D0940). Hydroxyphenyl Fluorescein can produce strong green fluorescence through hydroxyl radical reaction with intracellular peroxynitroso. Hydroxyphenyl Fluorescein can be applied for fluorescence microscopy, high-throughput imager, luciferase microplate reader or flow cytometry. Ex/Em=490/515 nm^[1].
Solvent and concentration: DMSO: 10 mM

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Preparation of HPF working solution
1.1 Stock solution
It is recommended to store the stock solution at -20°C -80°C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of HPF working solution
Dilute the stock solution in serum-free cell culture medium or PBS.The corresponding stock solution can be diluted according to the actual situation. Note that if the solvent is DMSO, the cytotoxicity of DMSO must be considered, and a solvent control should be prepared; if the solvent is pure water, the working solution needs to be filtered and sterilized before adding cells.
Note: Please adjust the concentration of HPF working solution according to the actual situation.

2. Cell staining
2.1 Cell preparation
For suspension cells: Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of HPF working solution, and then incubate at room temperature for 15-30 minutes.
2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope or flow cytometer.

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

424.41

C₂₆H₁₆O₆

359010-69-8

Liquid

Colorless to light yellow

OC1=CC=C(OC2=CC3=C(C4(C5=CC=C(C=C5O3)O)C6=C(C(O4)=O)C=CC=C6)C=C2)C=C1

Room temperature in continental US; may vary elsewhere.

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

• [1]. Ming-De Li, et al. 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.  [Content Brief]