Mitochondria staining kit

Manufactured by Merck Group

Sourced in United States, Italy, Germany

The Mitochondria Staining Kit is a laboratory product designed to visualize and study mitochondria, the powerhouses of cells. It provides a reliable and efficient method for staining and detecting mitochondria in various cell types. The kit includes all the necessary reagents and protocols for easy and consistent mitochondrial labeling.

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Spelling variants of this product

Isolated Mitochondria Staining kit (4 citations) JC-1 mitochondria staining kit (2 citations)

23 protocols using mitochondria staining kit

Mitochondrial Membrane Potential Assay

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Mitochondria Staining Kit (Sigma Aldrich, Milan, Italy) based on the dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolocarbocyanine iodide) was used to measure changes in the mitochondrial inner membrane electrochemical potential gradient (Δψ) [17 (link)].
Aggregated JC-1 red fluorescence, representing intact mitochondria, and monomeric JC-1 green fluorescence of the disrupted mitochondria were detected at 525 nm (excitation)/590 nm (emission) and 490 nm (excitation)/530 nm (emission), respectively (Fluoroskan Ascent fluorimeter, ThermoLabsystems). A drop in the red/green fluorescence intensity ratio indicates mitochondrial depolarization.

Chiaino E., Micucci M., Cosconati S., Novellino E., Budriesi R., Chiarini A, & Frosini M. (2020). Olive Leaves and Hibiscus Flowers Extracts-Based Preparation Protect Brain from Oxidative Stress-Induced Injury. Antioxidants, 9(9), 806.

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Mitochondrial Membrane Potential Analysis

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Mitochondrial membrane potential (MMP) changes appearing in the intrinsic pathway of mitochondria were spectrophotometrically detected. MMP was measured in a Spectramax, M5 fluorimetric device, at wavelengths of 490 nm (excitation), 530 nm(emission) according to the fluorometric Mitochondria Staining Kit (Sigma-Aldrich, Schnelldorf, Germany, CS0390) protocol.

Koyuncu I., Gonel A., Durgun M., Kocyigit A., Yuksekdag O, & Supuran C.T. (2018). Assessment of the antiproliferative and apoptotic roles of sulfonamide carbonic anhydrase IX inhibitors in HeLa cancer cell line. Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 75-86.

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Mitochondrial Dysfunction Assays in Neuronal Cells

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βNF (Cat# N3633), EtOH (Cat# E7023), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Cat# M2128), 1-methyl-4-phenylpyridinium iodide (MPP⁺; Cat# D048), mitochondria staining kit containing 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolocarbocyanine iodide (JC-1; Cat# CS0390), 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Cat# 287810), dimethyl sulfoxide (DMSO; Cat# D8418), and the materials used for cell culture were obtained from Sigma-Aldrich (Milan, Italy). The mitochondrial complex I activity kit (Cat# BVN-K520) was obtained from Vinci-Biochem (Florence, Italy). The Alexa fluor 488™-Annexin V/ propidium iodide (PI) double staining kit (Cat# V13245), the opti-MEM media (Cat# 11058021), and the Lipofectamine^® 2000 reagent (Cat# 11668019) were purchased from Life Technologies (Monza, Italy). The mitochondrial reporter (Ds-Red vector) and the plasmid containing the photo-activatable green fluorescent protein (PA-GFP) gene were kindly provided by Professor Richard Youle, NIH (Bethesda, MD, USA).

Fernandez-Abascal J., Chiaino E., Frosini M., Davey G.P, & Valoti M. (2020). β-Naphthoflavone and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration. International Journal of Molecular Sciences, 21(11), 3955.

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Mitochondrial Integrity Evaluation

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Quality of isolated mitochondria and the integrity of membrane were assessed using the JC-1 test by Mitochondria Staining Kit (CS0390, Sigma, USA).

Park Y.J., Heo J., Kim Y., Cho H., Shim M., Im K, & Lim W. (2023). Glucocorticoids alleviate particulate matter-induced COX-2 expression and mitochondrial dysfunction through the Bcl-2/GR complex in A549 cells. Scientific Reports, 13, 18884.

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Mitochondrial Membrane Potential Analysis

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The JC-1 mitochondrial membrane potential (MMP) was analyzed using a mitochondria staining kit (Cat No: CS0390, Sigma, St. Louis, MO, USA). Cells were collected by centrifugation and resuspended in a staining solution containing 200× JC-1 and 1× staining buffer. The suspension was incubated at 37 °C in a CO₂ incubator for 20 min. The stained cells were collected by centrifugation and washed once with 1× JC-1 staining buffer. After washing, the cells were centrifugated again and resuspended in 1 mL staining buffer. The fluorescence intensity was analyzed using a Guava® easyCyte™ flow cytometer (Merck, Kenilworth, NJ, USA).

Yang C., Kim H.S., Park S.J., Lee E.J., Kim S.I., Song G, & Lim W. (2019). Inhibition of miR-214-3p Aids in Preventing Epithelial Ovarian Cancer Malignancy by Increasing the Expression of LHX6. Cancers, 11(12), 1917.

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Probing Mitochondrial Changes in TNBC Cells

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Recently, the JC-1 assay has been applied as a reliable method for probing the mitochondrial transmembrane potential (ΔΨm) changes that occur in the very early stage of cell apoptosis [25 (link)]. Here, we used the JC-1 assay to analyze changes in the apoptosis of TNBC cells. The variation in ΔΨm was estimated using a mitochondria staining kit (Sigma-Aldrich, St. Louis, USA), which used JC-1. Briefly, 96-well black plates were used to seed 5 × 10³ cells per well. After 48 hours of treatment, JC-1 was used to cultivate the cells for 15 minutes. The excitation and emission wavelengths of the live cell bioimager were set at 490 and 530 nm, respectively, for the imagery of JC-1 monomers. The emission and excitation wavelengths were set at 590 and 525 nm, respectively, for the J-aggregates.

Zhang C., Yang Y., Yi L., Paizula X., Xu W, & Wu X. (2021). HOXD Antisense Growth-Associated Long Noncoding RNA Promotes Triple-Negative Breast Cancer Progression by Activating Wnt Signaling Pathway. Journal of Breast Cancer, 24(3), 315-329.

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Mitochondrial Membrane Potential Analysis

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The mitochondrial Δψm was examined after staining MOLM-13 cells with the cationic, lipophilic dye JC-1 contained in the Mitochondria Staining Kit (Sigma-Aldrich), which upon aggregation exhibits a fluorescence emission shift from 530 nm (green monomer) to 590 nm (red “J-aggregates”=healthy cells). The cells (1 × 10⁵/1 ml) were seeded in 6-well plates and treated with 1 ml of stenodactylin 10^-9 M for 24 h. Control samples were carried out adding 1 ml of complete medium. Subsequently, cells were stained with 500 µl of JC-1 dye (1:100 in RPMI) and incubated at room temperature in the dark for 10 min, as previously described (Polito et al., 2016c (link)). The cells were washed three times and observed under the Nikon Eclipse E600W fluorescence microscope.

Mercatelli D., Bortolotti M., Andresen V., Sulen A., Polito L., Gjertsen B.T, & Bolognesi A. (2020). Early Response to the Plant Toxin Stenodactylin in Acute Myeloid Leukemia Cells Involves Inflammatory and Apoptotic Signaling. Frontiers in Pharmacology, 11, 630.

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Mitochondrial Membrane Potential Assessment

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The alteration of MMP was detected using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). The colon cancer cell lines were incubated with polydatin for 24 h, were stained using a JC-1 dye for 20 min, and finally, they were rinsed before analysis. Fluorescence levels were evaluated by a flow cytometer (BD Accuri C6 Plus, BD Bioscience). The number of cells analyzed in each dot plot was 10,000, respectively. JC-1 measurement data were compensated according to the Accuri cytometer application note. The experiment was performed in triplicate.

Bae H., Lee W., Song J., Hong T., Kim M.H., Ham J., Song G, & Lim W. (2021). Polydatin Counteracts 5-Fluorouracil Resistance by Enhancing Apoptosis via Calcium Influx in Colon Cancer. Antioxidants, 10(9), 1477.

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Mitochondrial Membrane Potential Assay

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Changes in the JC-1 MMP were determined using a Mitochondria Staining Kit (Cat no: CS0390, Sigma). The cells were seeded in 6-well plates and incubated for 24 h in serum-free medium until they reached 70% confluency. Then, cells were treated with laminarin in a dose-dependent manner for 48 h at 37 °C in a CO₂ incubator. Supernatants were discarded, and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in a staining solution, which included 200 × JC-1 and 1 × staining buffer, and incubated at 37 °C in a CO₂ incubator for 20 min. The stained cells were collected by centrifugation and washed once with 1 × JC-1 staining buffer. The cells were then centrifuged once more and resuspended in 1 mL staining buffer. Fluorescence intensity was analyzed using a FACSCalibur (BD Biosciences).

Bae H., Song G., Lee J.Y., Hong T., Chang M.J, & Lim W. (2020). Laminarin-Derived from Brown Algae Suppresses the Growth of Ovarian Cancer Cells via Mitochondrial Dysfunction and ER Stress. Marine Drugs, 18(3), 152.

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Measurement of Mitochondrial Membrane Potential

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Change in ΔΨm was measured using the mitochondria staining kit (Sigma-Aldrich). Promastigotes were treated or not with 17.7 μM pterostilbene, 65 μM piceatannol, 95.5 μM polydatin and 65 μM oxyresveratrol for 48 h and then incubated with JC-1 (5 μg/mL) staining solution (prepared according to the manufacturer’s instructions) for 20 min at 37°C. ΔΨm was measured in 96-well opaque plates using 490/530 nm excitation/emission wavelengths for JC-1 monomers and 525/590 nm excitation/emission wavelengths for J-aggregates in a SpectraMax Paradigm (Molecular Devices), as described previously [18 (link)].

Passos C.L., Ferreira C., Soares D.C, & Saraiva E.M. (2015). Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs. PLoS ONE, 10(10), e0141778.

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