MitoDyes Red 594/615,IC-1562

Product Description

MitoDyes Red 594/615 is a superior red-orange fluorescent probe designed for high-performance mitochondrial imaging in live cells. It selectively accumulates in the mitochondrial inner membrane by utilizing the transmembrane potential, and is optimally excited using standard Texas Red® or mCherry filter sets.

This probe contains a mildly thiol-reactive chloromethyl moiety that reacts with free thiol groups of mitochondrial proteins, allowing the dye to be retained after aldehyde fixation and detergent permeabilization . Unlike traditional mitochondrial probes such as TMRM or Rhodamine 123, MitoDyes Red is retained in mitochondria even after membrane potential loss, making it suitable for experiments requiring cell fixation or involving agents that affect mitochondrial energy status

The staining procedure is simple and rapid: a short 15-minute incubation is sufficient to achieve stable, bright fluorescence signals, making it an ideal choice for both super-resolution and long-term live-cell imaging applications. Each vial provides sufficient material for 20 super-resolution imaging experiments .

This probe contains a mildly thiol-reactive chloromethyl moiety that reacts with free thiol groups of mitochondrial proteins, allowing the dye to be retained after fixation .

Key Features

- Excitation/Emission: 594 nm / 615 nm (peak maxima)

- Excellent retention after aldehyde fixation and detergent permeabilization

- Selective accumulation in mitochondria independent of membrane potential after binding

- Significantly reduced phototoxicity and markedly enhanced brightness compared to conventional dyes

- Simple 15-minute staining protocol for stable, bright fluorescence

- Compatible with fluorescence microscopy, confocal microscopy, SIM, FLIM, and flow cytometry

- Red-orange fluorescence enables multicolor labeling with green/far-red fluorophores

Size

20 assays;100assays

Storage and Stability

Form:Lyophilized powder.
Storage Conditions£º-20¡æ, desiccated, protected from light.

Stability£ºStable for up to 12 months.

DMSO stock solution£º-20¡æ, protected from light,Stable for 2 weeks; aliquot to avoid freeze-thaw cycles. 

Transportation: Stable at ambient temperature during routine shipping .

Preparation of Stock and Working Solutions

3.1 Stock Solution Preparation

1. Allow the vial to equilibrate to room temperature before opening .

2. Reconstitute the lyophilized powder in high-quality, anhydrous DMSO.

3. For a 20 assays vial, add 20ul of DMSO to obtain a stock solution .

4. Mix gently to ensure complete dissolution.

5. Critical: Aliquot the stock solution into single-use portions and store at -20¡æ protected from light . Avoid repeated freeze-thaw cycles.

3.2 Working Solution Preparation

Dilute the DMSO stock solution into pre-warmed (37¡æ) growth medium or an appropriate buffer (e.g., PBS, HBSS) to achieve the desired final concentration .

Recommended Concentration Ranges:

- Flow cytometry: typical dilution: 1:1,000 to 1:3,000 from stock

- Fluorescence microscopy: 1:1,000 to 1:5,000

- For fixed samples: 1:1,000 to 1:5,000

Note: Optimal concentration depends on cell type and application. Titrate the dye for each experimental system to determine the ideal concentration that provides sufficient signal-to-noise ratio without causing non-specific staining or toxicity .

Staining Protocols

4.1 General Guidelines

- Perform all incubations in the dark to prevent photobleaching .

- Pre-warm working solutions to 37¡æ before adding to cells .

- Optimal staining time is typically 15-45 minutes; the simple 15-minute protocol is sufficient for most applications .

- Higher concentrations and longer incubations may be required for thicker specimens or tissues.

4.2 Staining Live Cells

For Adherent Cells

1. Culture cells on sterile coverslips, chamber slides, or multi-well plates .

2. Remove growth medium and replace with pre-warmed (37¡æ) MitoDyes Red working solution .

3. Incubate for 15 minutes (or up to 45 minutes for thicker specimens) under normal cell culture conditions (37¡æ, 5% CO₂) .

4. Remove the staining solution and wash cells 2-3 times with fresh, pre-warmed medium or PBS (5 minutes per wash) .

5. Observe immediately, or proceed with fixation (Section 4.4) .

For Suspension Cells

1. Collect cells by centrifugation (400¡Ág for 3-4 minutes) .

2. Wash cells with PBS and resuspend in pre-warmed MitoDyes Red working solution at a density of 1¡Á10⁶ cells/mL .

3. Incubate for 15-45 minutes at 37¡æ protected from light .

4. Centrifuge (400¡Ág for 3-4 minutes) and remove supernatant .

5. Wash cells twice with PBS or fresh medium .

6. Resuspend in appropriate buffer for analysis .

4.3 Staining for Flow Cytometry

1. Prepare cells as described in Section 4.2.

2. Critical: For flow cytometry applications, use lower concentrations (1/3000) due to the higher sensitivity of flow cytometers compared to microscopes .

3. After staining and washing, resuspend cells in PBS or flow cytometry buffer .

4. Analyze using a flow cytometer equipped with a red laser (633-647 nm excitation) and appropriate emission filters (670/30 nm or similar) .

5. Controls: Include unstained cells and cells treated with a mitochondrial uncoupler (e.g., 50-100 µM CCCP or FCCP for 15-30 minutes before staining) as negative controls to confirm mitochondrial specificity .

4.4 Fixation and Permeabilization (Optional) :A key advantage of MitoDyes Red is its retention after fixation, enabling subsequent immunostaining procedures .

1. After live-cell staining (Section 4.2), wash cells with appropriate buffer .

2. Fix cells with pre-warmed (37¡æ) 2-4% formaldehyde solution (in PBS or culture medium) for 10-15 minutes at 37¡æ .- Alternative: Ice-cold 100% methanol for 15 minutes at -20¡æ .

3. Wash cells 3 times with PBS (5 minutes each wash) .

4. For permeabilization (optional): Incubate fixed cells with 0.2% Triton X-100 in PBS for 10 minutes, then wash with PBS .- Alternative:Ice-cold acetone for 5 minutes .

5. Proceed with immunostaining or other downstream applications .

Spectral Characteristics

Excitation maximum: 579-594 nm;Emission maximum:599-615 nm.

Recommended laser line: 561 nm (yellow), 594 nm (orange).

Recommended emission filter: 615/30 nm bandpass or 610 nm long-pass.

Color:Red-orange (appears red under fluorescence).

Filter compatibility Texas Red®, mCherry, Cy3.5 filter sets.

Troubleshooting Guide

Safety and Disclaimers

Safety Precautions

- DMSO is a skin penetrant; handle with appropriate personal protective equipment (lab coat, gloves, safety goggles) .

- This product contains material that may be harmful if ingested or inhaled.

- Dispose of all waste in accordance with local regulations.

Limited Use Disclaimer

This product is for research use only. It is not approved for human or veterinary use, diagnostic procedures, therapeutic applications, or clinical testing in humans or animals .

Warranty and Liability

This product is sold for research purposes only. There are no warranties, expressed or implied, including any warranty of merchantability or fitness for a particular purpose. The user assumes all responsibility for safe handling and use of this product consistent with all applicable safety standards.

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