This plan is based on literature, please modify it to meet your specific needs[1][2].
1. Preparation of Dye Working Solutions
(1) Preparation of Stock Solution: Dissolve Real Thiol in DMSO to prepare a 10mM stock solution.
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) Preparation of Working Solution: Use serum-free culture medium, such as OptiMEM, diluted to 1µM.
Note: Prepare the working solution fresh and store briefly at 4°C or on ice.
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 1µM Real Thiol working solution to resuspend the cells, and incubate at 37°C in the dark for 5-15min. 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 100μL of dye working solution to achieve a final concentration of 10μM 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 5-15min. 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, and observe by fluorescence microscope or flow cytometry.
4. Fluorescence detection: Fluorescence was measured using the 405nm and 488nm channels, and intracellular GSH levels were quantified by calculating the ratio of the fluorescence intensities from the two channels.
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.
References:
[1] Oberstadt M, Stieler J, Simpong DL, et al. TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole. Sci Rep. 2018;8(1):2248.
[2] Mendiola AS, Ryu JK, Bardehle S, et al. Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation. Nat Immunol. 2020;21(5):513-524. |
