BODIPY 493/503

This plan only provides a guide for bodipy 493/ 503, please modify it to meet your specific needs.
1. Preparation of staining solution
(1) Prepare stock solution: Dissolve BODIPY 493/503 in DMSO and prepare a stock solution with a concentration of 1-5mM.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium or PBS) and prepare a working solution with a concentration of 1-5 μM.
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 1 mL of BODIPY 493/503 working solution to resuspend the cells, and incubate at 37 °C 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 37 °C 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 light of BODIPY 493/503 is 493/503 nm respectively.

Precautions:
① It is recommended to set up a positive control. The cells in the control group are incubated with 30 μM oleic acid for 8 hours before subsequent experiments are performed;
② Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
③ For your safety and health, please wear a lab coat and disposable gloves.