MitoMark Red I

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

1. Prepare dyeing solution

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

(2) Dye working solution: Dilute the storage solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare a MitoMark Red I working solution with a concentration of 25-500 nM.

Notice:

① The commonly used working concentration range of MitoMark Red I is 25-500nM. It is recommended to use a relatively low concentration for staining. If you use a higher concentration, other cellular structures may be stained. In order to reduce false positives that may be caused by overloading the probe, it is recommended to use as low a concentration as possible without affecting the staining effect;

② For subsequent cell staining that requires fixation and permeabilization, it is recommended to use a working concentration range of 100-500nM;

③ Please adjust and optimize the concentration of the working fluid according to the actual situation, and prepare it now.

2. Cell suspension staining (taking a 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 15-45 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 100μL 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 Red I has a maximum excitation/emission wavelength of 578/599 nm.

Precautions:

① If subsequent steps such as ICC require permeabilization, it is recommended to use a buffer containing surfactant (such as Triton X-100) to permeabilize the cells. Cells can also be permeabilized with ice-cold acetone for 5 minutes and then washed with PBS (even if the cells are not subsequently labeled with other antibodies, this acetone permeabilization step may also improve the signal-to-noise ratio);

② If cells need to be fixed and stained, it is recommended to use freshly prepared preheated buffer or culture medium containing 2-4% paraformaldehyde for fixation;

③ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;

④ For your safety and health, please wear a lab coat and disposable gloves.

References:

[1]. K Gilmore, M Wilson. The use of chloromethyl-X-rosamine (Mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation. 1999 Aug 1;36(4):355-8. doi: 10.1002/(sici)1097-0320(19990801)36:4<355::aid-cyto11>3.0.co;2-9.