Mito-ID^® Membrane potential cytotoxicity kit for microplates

A Real-time Mitochondrial membrane Potential Assay with Superior Sensitivity



ENZ-51019-KP002	1 Kit	223.00 USD

10X more sensitive than JC-1 with superior aqueous solubility
Photostable dual-emission dye
Suitable for time-course studies evaluating intact and compromised mitochondria
No-wash/No-medium removal
Separate Mito-ID® assay is available for detection of mitochondrial mass
Detects toxicity at lower drug/dose concentrations
No solvent artifacts as those seen with JC-1 formulation
Suitable for high-throughput applications

The Mito-ID® Membrane Potential Cytotoxicity Kit measures fluctuations in mitochondrial membrane potential (MMP) utilizing a cationic dual-emission dye that exists as green fluorescent monomers in the cytosol, and accumulates as orange fluorescent J-aggregates in the mitochondria. Mitochondria having a low membrane potential will accumulate low concentrations of dye and will exhibit green fluorescence while more highly polarized mitochondria will exhibit orange-red fluorescence. Cells exhibit a shift from orange to green fluorescence as mitochondrial function becomes increasingly compromised.

more

Figure 1: Detect mitochondrial perturbations with 10 times more sensitivity than JC-1. Mitochondrial membrane potential (MMP) was evaluated in HeLa cells treated with CCCP using Mito-ID® dye (red) or JC-1 (blue). Using a conventional fluorescence microplate reader, MMP was shown to decrease with increasing CCCP concentration as indicated by a decrease in orange fluorescence. Improved aqueous solubility of the dye and no-wash protocol minimizes variability, leading to a higher Z-factor (> 0.9) than that obtained with JC-1.

Figure 2: Dual-emission probe monitors energetic status. Mito-ID® Membrane Potential dye is a dual-emission probe emitting in the FITC channel as green signal in the cytosol (A) and in the orange channel within the mitochondria (B). When a perturbing drug such as CCCP is added (C/D), the dye exists primarily as green-fluorescent monomers in the cytosol (C) and no longer exhibits orange fluorescence in the mitochondria (D).

Figure 3: Real-time detection of mitotoxicity in drug screening. Time-course study of mitochondrial membrane potential changes using a BioTek Synergy™ Mx fluorescence microplate reader. HeLa cells were incubated with Mito-ID® MP dye for 30 minutes at room temperature (no serum or media removal). Rotenone was added to achieve concentrations of 1 µM, 3 µM and 9 µM. Mito-ID® MP dye was shown to be responsive to rotenone, as demonstrated by a decrease in orange signal.

Please mouse over

Product Specification

Quantity:	For 2 x 96-well microplates

Quality Control:	A sample kit from each lot of Mito-ID^® Membrane Potential Cytotoxicity Kit is assayed using the microplate-based assay described in the manual. The Z’-factor from CCCP-treated cells is greater than 0.6.

Kit/Set Contains:	Mito-ID^® MP Detection Reagent, 200 μL CCCP Control, 100 μL 10X Assay Buffer 1: 2.5 mL 50X Assay Buffer 2: 0.5 mL

Application:	Mito-ID^® Membrane Potential Cytotoxicity Kit enables monitoring of mitochondrial potential changes using a simple fluorescence microplate reader.

Short Term Storage:	-20°C

Long Term Storage:	-80°C

Handling:	Avoid freeze/thaw cycles. Protect from light.

Background / Technical Information:	Mitochondria play a central role in cellular metabolism, bioenergetics, and apoptosis. Decreased mitochondrial function is known to be a major contributor to drug-associated toxicity in various organs. Growing FDA emphasis on evaluating the mitotoxic effects of drug candidates has increased the importance of determining such effects early in the drug development process.

General Literature References

High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells: A.J. Woollacott & P.B. Simpson; J. Biomol. Screen 6, 413 (2001), Abstract;

A New Method for the Cytofluorometric Analysis of Mitochondrial Membrane Potential Using the J-Aggregate Forming Lipophilic Cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1): A. Cossarizza, et al.; Biochem. Biophys. Res. Commun. 197, 40 (1993), Abstract;

Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate forming lipophilic cation JC-1: S.T. Smiley, et al.; PNAS 88, 3671 (1991), Full Text