MitoMark Green I

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

1. Prepare dyeing solution

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

(2) Dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare an MitoMark Green I working solution with a concentration of 20-200 nM.

Attention:

① It is recommended to use a relatively low concentration for staining. If a higher concentration is used, it may stain other cell structures. To reduce false positives caused by excessive probe loading, it is recommended to use a low concentration as much as possible without affecting the staining effect;

② If cells are incubated in a dye free medium after staining, attenuation of fluorescence signals and vacuolization of cells will be observed;

③ Please adjust and optimize the concentration of the working solution according to the actual situation, and prepare it as needed.

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 15-45minutes. 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 100 μL of 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 15-45 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. Observe using a fluorescence microscope or flow cytometer. MitoMark Green I has a maximum excitation/emission wavelength of 490/512 nm.

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]. 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.