The changes in mitochondrial membrane potential (MMP) in different lung cells (A549, Calu-3 and Beas-2B) when treated with different formulations of PTX and CUR (10 µM total concentration) were measured using the JC-10 mitochondrial membrane potential assay kit (MAK159), Sigma-Aldrich (Sydney, Australia). Before the treatment, approximately 5 × 104 cells per well were inoculated into a black 96-well plate and incubated overnight. This was followed with a treatment of CUR alone, PTX alone and a combination of CUR/PTX at varying ratios for 24 h. Cells without treatment and medium were used as a control in the study. After treatment, cells were washed with PBS twice and this was followed by staining with JC-10 dye as per the protocol supplied by the manufacturer. The intensity of fluorescence was measured by the (SpectraMax M2; Molecular devices, Sydney, Australia) microplate reader at λex = 490/λem = 525 nm for green fluorescence and λex = 540/λem = 590 nm for red fluorescence. The ratio between red and green fluorescence over control was taken as a percentage of MMP depletion. Three independent experiments were conducted for this purpose.
Jc 10 mitochondrial membrane potential assay kit
Manufactured by Merck Group
Sourced in Australia
The JC-10 mitochondrial membrane potential assay kit is a laboratory tool used to measure the mitochondrial membrane potential in cells. It provides a fluorometric method for detecting changes in mitochondrial membrane potential, which is an important indicator of cellular health and function.
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4 protocols using jc 10 mitochondrial membrane potential assay kit
1
Mitochondrial Membrane Potential in Lung Cells
2
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was assessed using a JC-10 Mitochondrial Membrane Potential Assay kit (Sigma; catalog # MAK159) according to the manufacturer's instructions. Δψm values were expressed in arbitrary units. FCCP (10 µM, 15 min), was added to cells to define the Δψm depolarized values.
3
Evaluating Mitochondrial Membrane Potential
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The M-HeLa
cells grown on cover glasses were treated with lead compound1 at concentrations of 10 and 25 μM and stained with
a fluorescent dye JC 10 in the dark for 15 min. Then, the samples
were washed with the buffer provided by the manufacturer as part of
the JC-10 Mitochondrial Membrane Potential Assay Kit (Sigma-Aldrich,
Merck). Confocal microscopy studies were carried out using a laser
confocal microscope LSM 780.
cells grown on cover glasses were treated with lead compound
a fluorescent dye JC 10 in the dark for 15 min. Then, the samples
were washed with the buffer provided by the manufacturer as part of
the JC-10 Mitochondrial Membrane Potential Assay Kit (Sigma-Aldrich,
Merck). Confocal microscopy studies were carried out using a laser
confocal microscope LSM 780.
4
Mitochondrial Membrane Potential Assay in M-HeLa Cells
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The M-HeLa cells were
detached using a mixture of trypsin–Versene (1:3), suspended
in PBS and centrifuged at 2000 rpm for 5 min, and then washed twice
with ice-cold PBS, followed by resuspension in PBS. Further, the samples
were added with 10 μg/mL of the JC-10 Mitochondrial Membrane
Potential Assay Kit (Sigma-Aldrich, Merck) and incubated at 37 °C
for 10 min. After the cells were rinsed three times and suspended
in PBS, JC-10 fluorescence was observed by flow cytometry.
detached using a mixture of trypsin–Versene (1:3), suspended
in PBS and centrifuged at 2000 rpm for 5 min, and then washed twice
with ice-cold PBS, followed by resuspension in PBS. Further, the samples
were added with 10 μg/mL of the JC-10 Mitochondrial Membrane
Potential Assay Kit (Sigma-Aldrich, Merck) and incubated at 37 °C
for 10 min. After the cells were rinsed three times and suspended
in PBS, JC-10 fluorescence was observed by flow cytometry.
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