Mitoscreen flow cytometry mitochondrial membrane potential detection kit

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The BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit is a laboratory tool designed to detect and measure the mitochondrial membrane potential in cells using flow cytometry. The kit provides the necessary reagents and protocols to enable this analysis.

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Flow cytometry (2933 citations) BD™ MitoScreen Kit (48 citations) BD MitoScreen (7 citations) Mitochondrial membrane potential detection kit (4 citations) BD Mitoscreen Mitochondrial Membrane Potential Detection Kit (2 citations) Mitochondrial membrane potential detection ( (2 citations)

6 protocols using mitoscreen flow cytometry mitochondrial membrane potential detection kit

Intracellular Calcium Signaling Assay

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The following reagents were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA): dimethylsulfoxide (DMSO), RPMI 1640 medium, fetal bovine serum, l-glutamine, penicillin, streptomycin, Dulbecco’s phosphate buffer saline, without calcium chloride and magnesium chloride (PBS), trypan blue solution, thapsigargin, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA). Vacuum tubes with K₃EDTA were purchased from Vacutainer Systems (Franklin Lake, NJ, USA). FLUO-4 AM was purchased Life Technologies (Carlsbad, CA, USA). MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit were purchased from BD Biosciences (San Diego, CA, USA).

Ribeiro D., Freitas M., Rocha S., Lima J.L., Carvalho F, & Fernandes E. (2018). Calcium Pathways in Human Neutrophils—The Extended Effects of Thapsigargin and ML-9. Cells, 7(11), 204.

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Evaluating Mitochondrial Membrane Potential

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Mitochondrial membrane potential, an indicator of apoptosis, was evaluated using the lipophilic fluorochrome JC-1 and BD^™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (Catalog No. 551302). Snail CNS and OT tissues were washed with isotonic tris EDTA buffer and homogenized in distilled water pH 7.5. Aliquots (100 μl) of each cell suspension were transferred into a sterile 15 ml polystyrene centrifuge tube. The samples were centrifuged at 350 g for 10 min, the supernatant was removed, and 500 μl JC-1 working solution was added to each cell pellet. Samples were incubated for 10–15 min at 37 °C in a CO₂ incubator. Cells were then washed three times using 1x assay buffer obtained with the kit (2 ml, 1 ml and 0.5 ml, respectively) for 5 min and immediately analyzed on an Accuri C6 flow cytometer (Becton Dickinson, Sunnyvale, CA, USA). JC-1 accumulates in the mitochondria in a potential-dependent manner and its increase forms JC-1 aggregates. Staining values were calculated as the percentage of the total number of cells counted. All experiments were repeated three times.

Habib M.R., Ghoname S.I., Ali R.E., Gad El-Karim R.M., Youssef A.A., Croll R.P, & Miller M.W. (2020). Biochemical and apoptotic changes in the nervous and ovotestis tissues of Biomphalaria alexandrina following infection with Schistosoma mansoni. Experimental parasitology, 213, 107887.

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Mitochondrial Membrane Potential Assessment by Flow Cytometry

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Mitochondrial potential was measured using a BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences) according to the manufacturer’s protocol. Briefly, HeLa cells were incubated for 18 h in a 6-cm culture plate and treated with metformin. The cells were then trypsinized and pelleted by centrifugation at 1000 rpm, after which the cells were resuspended in PBS and counted, which confirmed there were fewer than 1 × 10⁶ cells per ml. The cells were then stained with JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide) for 10–15 min at 37 °C in a CO₂ incubator. The fluorescence intensity of the JC-1 was evaluated flow cytometrically. The excitation wavelength was 488 nm, while emission wavelengths of 530 nm (FL1-H channel) and 580 nm (FL2-H channel) were used to detect the JC-1 monomer and aggregates, respectively.

Hsieh Li S.M., Liu S.T., Chang Y.L., Ho C.L, & Huang S.M. (2018). Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1. Journal of Biomedical Science, 25, 81.

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Mitochondrial Membrane Potential Analysis

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Mitochondrial membrane potential was measured using a BD MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. The procedural details were described in our previous publications [33 (link),34 (link)]. All dead and viable cells were harvested, washed with PBS, and incubated with a 1× binding buffer containing the MMP-sensitive fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolyl carbo-cyanine iodide) for 15 min at 37 °C in the dark. The cells were then washed, and JC-1 fluorescence was measured in a flow cytometer with excitation at 488 nm and emission at 530 nm (FL1-H channel for green fluorescence) for the monomer and 580 nm (FL2-H channel for red fluorescence) for aggregates, with a FACSCalibur flow cytometer using Cell Quest Pro software (BD Biosciences, Franklin Lakes, NJ, USA). The mitochondrial depolarization was measured as a function of the decrease in the red/green fluorescence intensity ratio.

Lin C.J., Chen J.T., Yeh L.J., Yang R.C., Huang S.M, & Chen T.W. (2022). Characteristics of the Cytotoxicity of Taraxacum mongolicum and Taraxacum formosanum in Human Breast Cancer Cells. International Journal of Molecular Sciences, 23(19), 11918.

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Mitochondrial Membrane Potential Assay

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The mitochondrial potential was performed using BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences), according to the manufacturer’s instructions. In brief, HeLa cells were seeded on a 6 cm culture plate and incubated with the amiodarone dose. After 18 h treatment, cells were trypsinized and collected cell pellets at 1000 rpm, resuspended the cells in PBS and to count cell number, cell counting should not exceed 1 × 10⁶ cells per ml. All the treated cells were the stained with the JC-1 dye for 10∼15 min at 37° C in a CO₂ incubator. After washing twice with 1x Assay Buffer, the pellets were resuspended in 1x Assay Buffer (0.5 ml) and the fluorescence of the JC-1 dye was measured by flow cytometric analysis by exciting the dye at 488 nm and detecting the JC-1 monomer through its emission at 530 nm (FL1 channel) with aggregates of JC-1 being detected at 580 nm (FL2 channel).

Chang Y.L., Liu S.T., Wang Y.W., Lin W.S, & Huang S.M. (2018). Amiodarone promotes cancer cell death through elevated truncated SRSF3 and downregulation of miR-224. Oncotarget, 9(17), 13390-13406.

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Measurement of Mitochondrial Membrane Potential

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Mitochondrial potential was measured using a BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences) according to the manufacturer’s protocol. Briefly, the cells were exposed to siGRPEL2 for 48 h in 6-well plates. The cells were then trypsinized and pelleted by centrifugation at 1000 rpm, after which the cells were resuspended in PBS and counted, which confirmed that there were fewer than 1 × 10⁶ cells per mL. The cells were then stained with JC-1 dye (5,5′,6,6′- tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide) for 10–15 min at 37 °C in a CO₂ incubator. The fluorescence intensity of the JC-1 was evaluated by flow cytometry. The excitation wavelength was 488 nm, while emission wavelengths of 530 nm (FL1-H channel) and 580 nm (FL2-H channel) were used to detect the JC-1 monomer and aggregates, respectively.

Tang C.T., Li Y.F., Chou C.H., Huang L.C., Huang S.M., Hueng D.Y., Tsai C.K, & Chen Y.H. (2021). GRPEL2 Knockdown Exerts Redox Regulation in Glioblastoma. International Journal of Molecular Sciences, 22(23), 12705.

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