MitoBrilliant™ Protocol
Next-generation fluorescent stains for the localization and tracking of mitochondria
ProductUnitSize

MitoBrilliant™ 646

Abs/Em(nm)Extinction
CoefficientΔψm
Dependent?

Stain pre-fixedcells?

(M-1 cm-1)

5x50 µg655/668

125,000

No*Yes5x50 µg648/662

127,800

Yes

No

5x50 µg

550/568

100,000

Yes

No

Cat. No. 7700

MitoBrilliant™ Live 646
Cat. No. 7417

MitoBrilliant™ Live 549
Cat. No. 7693

* 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 Product Guide.

Storage and HandlingStore at -20°C and protect from light. We recommend that stock solutions, once prepared, are storedaliquoted in tightly sealed vials at -20°C or below and used within 1 month.

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Reconstitution and Stock Solution Preparation
Prepare a 1mM stock solution by adding high quality, anhydrous DMSO to one vial of MitoBrilliant™ dye.
ProductMitoBrilliant™ 646

Molecular Weight Quantity per vial

Recommended reconstitution

493.650 µg (101 nmoles)

Add 101 µL DMSO for a 1 mM stock solution

445.150 µg (112 nmoles)

Add 112 µL DMSO for a 1 mM stock solution

417.0

50 µg (120 nmoles)

Add 120 µL DMSO for a 1 mM stock solution

Cat. No. 7700

MitoBrilliant™ Live
646
Cat. No. 7417
MitoBrilliant™ Live
549
Cat. No. 7693

Staining Live Cells
1. 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 finalconcentration used for individual experiments. Aqueous working solutions should be prepared andused on the same day.
2. Incubate for 30 to 60 minutes at 37°C prior to imaging (longer incubation times may give brighterstaining). 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.

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Staining Cells in Suspension for Flow Cytometry
1. 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.
2. Incubate for 30-60 minutes at 37°C in the dark.
3. 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 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 optimalstaining. 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 berequired 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.

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MitoBrilliant™ Live 549~800CQ):g60Q)

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Global info@bio-techne.com bio-techne.com/find-us/distributors TE L + 1 612 379 2956 North America TE L 800 343 7475
Europe I Middle East I Africa TEL +44 (0)1235 529449 China info.cn@bio-techne.com TEL +86 (21) 52380373

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Trademarks and registered trademarks are the property of their respective owners.