MitoBrilliant™ Protocol
This is intended as a guide only; for full experimental details please read the MitoBrilliant Product Guide provided.
In Brief Download the PDF of this protocol
Next-generation fluorescent stains for the localization and tracking of mitochondria
| Product | Unit Size | Abs/Em (nm) | Extinction Coefficient (M-1 cm-1) | Δψm Dependent? | Stain pre-fixed cells? |
|---|---|---|---|---|---|
| MitoBrilliant™ 646 Cat. No. 7700 | 5x50 µg | 655/668 | 125,000 | No* | Yes |
| MitoBrilliant™ Live 646 Cat. No. 7417 | 5x50 µg | 648/662 | 127,800 | Yes | No |
| MitoBrilliant™ Live 549 Cat. No. 7693 | 5x50 µg | 550/568 | 100,000 | Yes | No |
* In live-cell staining, the mitochondrial membrane potential (Δψm) helps to drive initial recruitment of the dye into mitochondria. After staining, localization of the dye becomes insensitive to Δψm changes. In pre-fixed cell staining, MitoBrilliant™ 646 can localize and be retained in mitochondria without Δψm. For more information on MitoBrilliant™ including application data, please refer to the MitoBrilliant Product Guide.
Storage and Handling
Store at -20°C and protect from light. We recommend that stock solutions, once prepared, are stored aliquoted in tightly sealed vials at -20°C or below and used within 1 month.
Reconstitution and Stock Solution Preparation
Prepare a 1mM stock solution by adding high quality, anhydrous DMSO to one vial of MitoBrilliant™ dye. For more information on preparing stock solutions and reconstitution, please see our Molarity Calculator and Reconstitution Calculator.
| Product | Molecular Weight | Quantity per vial | Recommended reconstitution |
|---|---|---|---|
| MitoBrilliant™ 646 Cat. No. 7700 | 493.6 | 50 µg (101 nmoles) | Add 101 µL DMSO for a 1 mM stock solution |
| MitoBrilliant™ Live 646 Cat. No. 7417 | 445.1 | 50 µg (112 nmoles) | Add 112 µL DMSO for a 1 mM stock solution |
| MitoBrilliant™ Live 549 Cat. No. 7693 | 417.0 | 50 µg (120 nmoles) | Add 120 µL DMSO for a 1 mM stock solution |
Staining Live Cells
- Dilute the 1mM DMSO stock solution using a warm (37 °C) buffer or growth medium and apply to live cells at a final working concentration between 50 to 200 nM. We recommend optimizing the final concentration used for individual experiments. Aqueous working solutions should be prepared and used on the same day.
- Incubate for 30 to 60 minutes at 37°C prior to imaging (longer incubation times may give brighter staining). A washing step prior to imaging is not required but it is recommended: rinse the cells with 1x PBS and apply fresh media prior to imaging.
Staining Cells in Suspension for Flow Cytometry
- After gentle centrifugation, carefully resuspend the cell pellet in a prewarmed (37°C) buffer or growth medium containing 50-200 nM MitoBrilliant™ at a cell concentration of 1 x 106 per mL.
- Incubate for 30-60 minutes at 37°C in the dark.
- Re-pellet the cells buffer by gentle centrifugation, then resuspend cells in fresh pre-warmed medium ready for flow cytometry.
Staining Fixed Cells - Only Applicable to: MitoBrilliant™ 646 (Cat. No. 7700)
MitoBrilliant™ 646 can be applied at either the pre-fixation or post-fixation stages of sample preparation. For more application information, please refer to the MitoBrilliant Product Guide.
- Dilute the 1 mM DMSO stock solution to a final working concentration between 50 to 200 nM. We recommend optimizing the final concentration used for individual experiments. Aqueous working solutions should be prepared and used on the same day.
- Appropriate fixation methods are critical for preserving cellular structure and achieving optimal staining. For HeLa cells, we recommend fixing with freshly prepared pre-warmed buffer or growth medium containing 4% paraformaldehyde at 37 °C for 10 to 20 minutes. Optimization might be required for different model systems. After fixation, rinse the cells several times in PBS.
- Permeabilization step (optional). Incubate fixed cells for 10 minutes in PBS containing 0.05% Triton® X-100.
