1,3-Diphenylisobenzofuran

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare dyeing solution
(1) Prepare dye stock solution: Use high-quality anhydrous DMSO to prepare a stock solution with a concentration of 10-20mM;
Note: It is recommended to aliquot unused stock solution and store it in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare dye working solution: Use an appropriate buffer (such as serum-free cell culture medium or PBS, which needs to be preheated) to dilute the stock solution and prepare a dye working solution with a concentration of 10-20 μM.
Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.
2. Cell staining
(1) Suspension cells: centrifuge at 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS or other buffer, 5 minutes each time;
(2) Adherent cells: wash twice with PBS or other buffers, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed;
(3) Add dye working solution to resuspend the cells, and incubate the cells in the dark at room temperature for 15-45 minutes to allow the dye to penetrate;
Notice:
① It is recommended to resuspend the cells every 5-10 minutes during the staining process to ensure full staining;
② 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 to remove the staining solution, add PBS or other buffers and wash 2-3 times to remove residual probes.
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 about 100-150μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye;
(4) Incubate the cells at room temperature in the dark for 15-45 minutes to allow dye penetration;
Note: 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 wash the coverslip 2 to 3 times with PBS or other buffers.
4. Fluorescence detection: Observe the staining results through a fluorescence microscope or flow cytometer. The maximum excitation/emission wavelength of 1,3-Diphenylisobenzofuran is 495/523nm.

Precautions:
1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.