Fluorescent dye
MitoTracker Deep Red FM targets mitochondria in live cells. The dye does not target a specific protein or enzyme but instead accumulates in active mitochondria due to their membrane potential. Upon accumulation in the mitochondrial matrix, the dye covalently binds to mitochondrial proteins through reaction with free thiol groups of cysteine residues. This covalent binding allows the dye to be retained in mitochondria even after the loss of membrane potential, enabling the study of mitochondrial morphology and dynamics independently of membrane potential.

MitoTracker Deep Red FM Usage Guide
I. Working Solution Preparation
1. Stock Solution Preparation
Dissolve MitoTracker Deep Red FM in DMSO to prepare a 1 mM stock solution. For example: Add 50 μg of the reagent into 92 μL of DMSO for dissolution.
Note: The stock solution should be aliquoted and stored protected from light at -20°C or -80°C.

2. Working Solution Preparation
Dilute the stock solution at a ratio of 1:5000 to 1:50000 using pre-warmed serum-free cell culture medium or PBS to obtain a 20–200 nM staining working solution.
Recommendation: The working solution concentration can be optimized based on experimental conditions and should be prepared immediately before use.

II. Cell Staining Procedure
1. Staining of Suspension Cells
- Collect cells, wash twice with PBS (5 minutes each time), and adjust the cell density to 1×10⁶/mL;
- Add 1 mL of working solution and incubate at room temperature protected from light for 15–45 minutes;
- Centrifuge at 400 g for 3–4 minutes and remove the supernatant;
- Wash twice with PBS;
- Resuspend the cells in serum-free medium or PBS, then proceed with observation using a fluorescence microscope or flow cytometry.

2. Staining of Adherent Cells
- Culture cells on sterile coverslips, and remove the medium before staining;
- Add 100 μL of working solution, ensuring complete coverage of the cell layer, and incubate at room temperature protected from light for 15–45 minutes;
- Remove the working solution and wash 2–3 times with fresh medium (5 minutes each time);
- Proceed directly with fluorescence observation.

III. Storage Conditions
Store protected from light at -20°C for up to one year.

IV. Precautions
- The stock solution should be aliquoted, stored protected from light, and avoided repeated freeze-thaw cycles. It can be stored stably at -20°C for approximately one month, and at -80°C for up to six months;
- The working solution concentration should be optimized according to the actual cell type and staining effect;
- This product is intended for research use only and is not suitable for clinical diagnosis or treatment, food, or drug applications;
- Wear lab coats and disposable gloves during operation to ensure safety.

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The in vitro activity of MitoTracker Deep Red FM is its ability to selectively stain mitochondria in live cells. The dye exhibits bright far-red fluorescence, with excitation/emission maxima at 644/665 nm, providing excellent signal-to-noise ratio and minimal background autofluorescence. The staining is dependent on mitochondrial membrane potential, with active mitochondria accumulating more dye. The dye is cell-permeable and can be used at low nanomolar concentrations to minimize cellular toxicity. It enables visualization and analysis of mitochondrial morphology, distribution, and dynamics. MitoTracker Deep Red FM is compatible with multiplexing with other fluorescent dyes due to its far-red emission spectrum.

No significant in vivo pharmacological activity has been reported for MitoTracker Deep Red FM, as the compound is primarily utilized as a research tool for mitochondrial staining rather than as a therapeutic agent. The dye is not intended for systemic therapeutic use.

In vitro binding assays are not applicable for MitoTracker Deep Red FM as it functions as a mitochondrial dye rather than a drug targeting specific enzymes or receptors. The compound's interaction with mitochondria is based on its membrane potential-dependent accumulation and covalent binding to mitochondrial proteins.

For in vitro cellular experiments, MitoTracker Deep Red FM is dissolved in DMSO to prepare stock solutions and diluted in cell culture medium at recommended concentrations (typically 50-200 nM). Cells are incubated with the dye at 37°C for 15-45 minutes, allowing the dye to accumulate in active mitochondria. After incubation, cells are washed with fresh medium to remove excess dye and analyzed by fluorescence microscopy or flow cytometry. The far-red fluorescence is excited with a 633-644 nm laser and detected at 665-750 nm. The dye is compatible with live-cell imaging and can be used for time-lapse studies of mitochondrial dynamics.

For in vivo animal experiments, MitoTracker Deep Red FM is not typically used as a systemic imaging agent. However, the dye can be used for ex vivo analysis of mitochondria in tissues harvested from animal models. The dye may also be used for staining mitochondria in tissue sections or isolated mitochondria preparations.

Pharmacokinetic properties of MitoTracker Deep Red FM have not been characterized, as the compound is a research-use fluorescent dye rather than a drug candidate. The compound should be stored as powder at -20°C for up to 3 years or in solution at -20°C for up to 6 months, protected from light.

The dye is intended for research use only and not for human therapeutic or diagnostic applications. Standard laboratory safety precautions should be observed when handling this chemical reagent, including protection from light.

[1]. MitoTracker labeling in primary neuronal and astrocytic cultures: influence of mitochondrial membrane potential and oxidants. J Neurosci Methods. 2001 Jan 15;104(2):165-76.

MitoTracker Deep Red FM is a far-red fluorescent dye that selectively stains mitochondria in live cells. The dye accumulates in active mitochondria dependent on membrane potential and covalently binds to mitochondrial proteins. It is widely used in cell biology research to study mitochondrial morphology, distribution, and dynamics. The dye is compatible with live-cell imaging, flow cytometry, and multiplexing with other fluorescent probes. It has no clinical or therapeutic applications and has not received regulatory approval.

C34H36CL2N2

543.569046974182

873315-86-7

Blue to dark blue solid powder

ClCC1C=CC(=CC=1)CN1C2C=CC=CC=2C(C)(C)/C/1=C\C=C\C=C\C1C(C)(C)C2C=CC=CC=2[N+]=1C.[Cl-]

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 125 mg/mL (229.96 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)