Mito-Tracker Green is a green fluorescent dye that selectively accumulates in the mitochondrial matrix. MitoTracker Green FM covalently binds mitochondrial proteins by reacting with free mercaptan of cysteine residues, allowing staining of mitochondrial membrane potential independent of membrane potential. Excitation/emission wavelength 490/523 nm.

General Protocol

1. Preparation of MitoTracker Green FM working solution
1.1 Preparation of the stock solution
Dissolve 50 μg MitoTracker Green FM in 74.4 μL DMSO to obtain 1 mM of stock solution.
Note: It is recommended to store the stock solution at -20°C and -80°C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of MitoTracker Green FM working solution
Dilute the stock solution in serum-free cell culture medium or PBS dilute at 1:5000 or 1:50000 to obtain 20-200 nM of working solution.
Note: Please adjust the concentration of MitoTracker Green FM working solution according to the actual situation.
2. Cell staining
2.1 Suspension cells（6-well plate）
a. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×10⁶/mL.
b. Add 1 mL of working solution, and then incubate at room temperature for 15-45 minutes.
c. Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 5-30 minutes.
d. Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.

Storage
-20°C, 1 year. Protect from light

Precautions
1. Please adjust the concentration of MitoTracker Green FM working solution according to the actual situation.
2. This product is for R&D use only, not for drug, household, or other uses.
3. For your safety and health, please wear a lab coat and disposable gloves to operate.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Gautam N, et, al. A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization. Methods. 2018 Feb 1;134-135:11-19.   [Content Brief]

  [2]. Gautam N, et, al. A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization. Methods. 2018 Feb 1;134-135:11-19.  [Content Brief]