HPF (Synonyms: Hydroxyphenyl Fluorescein)

This plan only provides a guide, please modify it to meet your specific needs.

1. Preparation of staining solution

(1) Prepare stock solution: Dissolve HPF in DMSO and prepare a stock solution with a concentration of 1-10mM.

Note:

① HPF can be dissolved in DMSO, but data shows that DMSO is a quencher for hydroxyl radicals, which will interfere with detection and reduce sensitivity. It is recommended to use DMF as a solvent;

② Compared with DMSO, DMF has relatively high cytotoxicity. It is recommended that customers use a lower concentration of HPF under the premise of achieving the ideal fluorescence effect, and verify the sensitivity of the cells to the corresponding dose of solvent before the experiment;

③ Aliquot unused storage solution and store in the dark at -20℃ or -80℃ to avoid repeated freezing and thawing.

(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as HHBS or PBS) and prepare a working solution with a concentration of 1-10μM.

Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.

2. Cell suspension staining

(1) Suspension cells: Centrifuge at 1000g for 3-5 minutes at 4°C, discard the supernatant, and wash twice with PBS for 5 minutes each time.

(2) Adherent cells: Wash twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(3) Add HPF working solution to resuspend the cells, and incubate at room temperature in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.

(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.

(5) Resuspend cells in pre-warmed serum-free cell culture medium or PBS and observe by fluorescence microscope or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.

(3) Add 100 μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.

(4) Incubate at room temperature in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.

(5) After the incubation, discard the dye working solution and use pre-warmed culture solution to wash the coverslip 2 to 3 times.

4. Microscope detection: The maximum excitation/emission wavelength of HPF is 490/515nm.

Precautions:

① BSA and phenol red will affect the fluorescence of HPF. It is recommended to use a buffer system or culture medium that does not contain BSA and phenol red for experiments;

② HPF can be used for solution system or intracellular ROS detection. The recommended starting working concentration is 1-10 μM. Please optimize according to your personal experimental system or reference literature;

③ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;

④ For your safety and health, please wear a lab coat and disposable gloves.