Computational protocol: Scutellaria baicalensis Extracts and Flavonoids Protect Rat L6 Cells from Antimycin A-Induced Mitochondrial Dysfunction

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Protocol publication

[…] To detect changes in MMP, JC-1 was used as an indicator of mitochondrial function. The dye 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluoresces red or green, respectively, when it aggregates in healthy mitochondria with high membrane potentials or exists as a monomer in mitochondria with diminished membrane potential. Cells were seeded in 96-well plates at 1 × 104 cells/well. MMR was measured using a JC-1 mitochondrial membrane potential detection kit []. Before adding JC-1 the medium was aspirated from the plates and adherent cells washed with PBS. The plates were incubated at 37°C for 20 min after the addition of 100 μL of 1× JC-1 reagent into the wells. Cells were washed twice with PBS, and then PBS was added in an amount sufficient to cover the cell layer. Red fluorescence (excitation, 550 nm, and emission, 600 nm) and green fluorescence (excitation, 485 nm, and emission, 535 nm) were determined using a Softmax Pro fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). The ratio of red-to-green fluorescence in dead cells and in cells undergoing apoptosis is decreased compared with healthy cells. For confocal microscopy, 1× of JC-1 was added to treated cells for 15 min at 37°C. Cells were imaged using Olympus FV10i-LIV confocal microscopes (Olympus, Tokyo, Japan). In live nonapoptotic cells, mitochondria appeared red due to aggregation of the JC-1 reagent. The red aggregates are excited at 559 nm and emit at 570–620 nm. In apoptotic and dead cells, the dye is monomeric and emits at 490–540 nm when excited at 473 nm. [...] Cells were incubated with varying concentrations of extracts and 100 μg/mL of AMA harvested and lysed in RIPA buffer (T&I, Seoul, Korea). Protein concentrations were determined using the Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was incubated with the primary antibody (1 : 1000) overnight and the secondary antibody (1 : 5000) for 2 h. The blot was then developed using Luminol Enhancer solution (GE Healthcare, Waukesha, USA), visualized using an ImageQuant LAS 4000 mini (GE Healthcare, Waukesha, USA), and quantified by using Image J densitometry software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,, 1997–2011). […]

Pipeline specifications

Software tools SoftMax Pro, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Scutellaria baicalensis, Rattus norvegicus, Leuciscus baicalensis
Diseases Mitochondrial Diseases
Chemicals Adenosine Triphosphate, Antimycin A, Flavonoids, Oxygen, Superoxides