Hydroxyphenyl Fluorescein

Hydroxyphenyl Fluorescein (HPF) 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]. In Vitro:Preparation of HPF working solution
1.1 Preparation of the stock solution
Dissolve 1 mg of HPF to obtain 10 mM of HPF.
Note: 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 to obtain 1-10 μM of HPF working solution.
Note: Please adjust the concentration of HPF working solution according to the actual situation.

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 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.