This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare dyeing solution
(1) Prepare stock solution: Add 53μl anhydrous DMSO to a tube containing 50μg Mito-FerroGreen, pipet and mix with a pipette to prepare a Mito-FerroGreen stock solution with a concentration of 1mM.
(2) Prepare working solution: Use serum-free culture medium or HBSS buffer to dilute Mito-FerroGreen stock solution and prepare Mito-FerroGreen working solution with a concentration of 5μM.
Notice:
①Neither the 1 mM Mito-FerroGreen stock solution nor the 5μM Mito-FerroGreen working solution can be stored for a long time, and it is recommended to prepare it for immediate use;
②Since the degradation of Mito-FerroGreen will increase the background fluorescence, if it cannot be used up at one time, it is not recommended to continue to use the remaining Mito-FerroGreen stock solution and working solution;
③Ethanol can be used instead of DMSO. The working solution prepared with ethanol can be stored stably for 2 weeks at -20°C;
④Do not use serum-containing culture media when preparing working solutions. Serum will cause excessive staining background.
2. Cell staining
(1) After inoculating cells on a culture dish/copolymer dish/sterile coverslip, culture in a 37°C, 5% CO2 incubator overnight or until the cells are completely attached;
(2) After removing the medium, wash the cells three times with serum-free medium or HBSS buffer;
(3) Add Mito-FerroGreen working solution with a concentration of 5μM, and incubate for 30 minutes at 37°C and 5% CO2;
(4) Remove the supernatant and wash the cells three times with serum-free medium or HBSS buffer.
(5) (optional, positive control group) Add culture medium containing activators (such as Erastin, RSL3), and culture in a 37°C, 5% CO2 incubator for 1 hour;
Notice:
①Please optimize the specific culture time according to the experimental conditions;
②The order of steps (3) and (5) can be changed according to specific experimental conditions.
3.Observe cells under a fluorescence microscope. The maximum absorption/emission wavelength of Mito-FerroGreen is 505/535nm.
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.