Mito-Tracker Deep Red 633

Instructions for Use:

1. Preparation of Mito-Tracker Deep Red 633 stock solution.

After equilibrating a tube of 50μg Mito-Tracker Deep Red 633 powder to room temperature, add 426μl of anhydrous DMSO (anhydrous dimethylsulfoxide), and dissolve thoroughly to obtain 0.2mM Mito-Tracker Deep Red 633 stock solution. Store at -20℃ or lower temperatures after proper dispensing and protected from light.

2. Preparation of Mito-Tracker Deep Red 633 staining solution.

a. Add 0.2mM Mito-Tracker Deep Red 633 stock solution to cell culture medium or an appropriate solution (e.g., HBSS with calcium and magnesium ions) at a ratio of 1:4000-1:1000 to a final concentration of 50nM-200nM. For example, take 1µl of 0.2mM Mito-Tracker Deep Red 633 and add it to 4ml or 1ml of cell culture medium, mix well, and then it will be the Mito-Tracker Deep Red 633 staining solution, with a final concentration of 50nM or 200nM, respectively.

b. Mito-Tracker Deep Red 633 staining solution should be pre-warmed at 37℃ before use.

Note: The concentration of staining solution can be adjusted appropriately according to the actual situation. To reduce non-specific fluorescent staining and mitochondrial toxicity, we recommend using a low concentration.

3. Mitochondrial staining of adherent cells.

a. When the cells are incubated to a certain density in cell culture plates or Petri dishes, remove the cell culture medium, add the Mito-Tracker Deep Red 633 staining solution prepared in step 2, and incubate at 37℃ for 15-30 minutes.

Note: The optimal incubation time should be optimized appropriately according to the cell type.

b. Remove the Mito-Tracker Deep Red 633 staining solution and add fresh cell culture medium pre-incubated at 37℃.

c. Observe with a fluorescence microscope, laser confocal microscope, or fluorescence microplate reader. If the staining is not satisfactory, the concentration of Mito-Tracker Deep Red 633 staining solution can be increased or the staining time can be extended appropriately within the recommended range.

Note: Mito-Tracker Deep Red 633 is prone to quenching, so please perform fluorescence assays or take pictures as soon as possible. Quenching can also be delayed by reducing the excitation intensity of the fluorescence microscope (i.e. mercury lamp or LED light source) or by appropriately reducing the concentration of Mito-Tracker Deep Red 633 staining solution.

4. Mitochondrial staining of suspension cells.

a. Centrifuge at 1000g for 5 min, discard the supernatant, gently resuspend cells with 37℃ pre-warmed Mito-Tracker Deep Red 633 staining solution and incubate at 37℃ for 15-30 min.

Note: The optimal incubation time should be optimized appropriately according to cell type.

b. After incubation, centrifuge at 1000g for 5 minutes, discard the supernatant and resuspend the cells by adding fresh cell culture medium pre-incubated at 37℃.

c. Observe with a fluorescence microscope, laser confocal microscope, flow cytometer, or fluorescence microplate reader.

Note: If suspension cells need to be fixed on a slide or coverslip, the slide or coverslip can first be treated with Poly-D-lysine.