TMRM

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

1. Prepare TMRM staining solution

(1) Place the cryopreserved TMRM powder at room temperature for at least 20 minutes (this step is very important, do not ignore the operation), and centrifuge at low speed for a moment to allow the powder to sink to the bottom of the tube.

(2) Dye stock solution: Use DMSO to dissolve TMRM into a 1-5mM stock solution. The prepared stock solution is aliquoted and stored in the dark at -20 or -80°C.

(3) Dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare a TMRM working solution with a concentration of 20nM~1μM.

Notice:

① Since the usage concentration of TMRM is relatively low, it is recommended to use DMSO to dilute and prepare an intermediate storage concentration (for example: 100uM);

② Please adjust and optimize the working fluid concentration according to the actual situation, and prepare it now.

2. Cell suspension staining (taking 6-well plate as an example)

(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.

(2) Wash the adherent cells 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 dye working solution to resuspend the cells, and incubate at room temperature in the dark for 5-30 minutes. The optimal culture time for different cells is different.

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

(5) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

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

(3) Add 100uL dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 30-60 minutes.

(4) Aspirate away the dye working solution and use culture solution to wash the coverslip 2 to 3 times for 5 minutes each time.

4. Microscope detection: TMRM has two excitation peaks at 515 and 552 nm, and the maximum excitation light/emission light is 552/575nm.

Precautions:

①For fluorescence imaging analysis, the commonly used TMRM working concentration range is 50-200nM;

② If the effect of a certain drug on membrane potential needs to be measured, the cells need to be processed in advance, and then the probe is used for subsequent detection. At the same time, a drug-untreated group and a drug-treated group need to be set;

③ If you need a positive control, you can choose FCCP (Cat. No.: GC14328) or CCCP (Cat. No.: GC14727). Both compounds are uncoupling agents that can reduce the mitochondrial membrane potential, thereby preventing TMRM staining;

④ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;

⑤ For your safety and health, please wear a lab coat and disposable gloves.References:

[1]. Michael B Schultz,et. Molecular and Cellular Characterization of SIRT1 Allosteric Activators. 2019:1983:133-149. doi: 10.1007/978-1-4939-9434-2_8.