Jc 1 staining kit

Mitochondrial membrane potential (MMP) was measured by JC-1 staining kit (Cat. no. CS0390, Sigma) following manufacturer's instructions. 0.2 × 10⁶ THP-1 cells were seeded in 24 well plates and cultured overnight in complete medium. At the end of the experiment, cells from the different experimental categories described earlier were assayed. Briefly, the cells were washed with PBS and stained with 2.5 μM of JC-1 in 1X JC-1 staining buffer. These cells were further incubated for 20 min at 37°C in a humidified atmosphere containing 5% CO_2. Further, the cells were washed with ice-cold 1X JC-1 staining buffer. The cells were then resuspended in 0.5 ml of the same buffer and analysis of the samples was performed using spectrofluorimetry (spectramax m5). JC-1 monomers were detected in the FL1 channel (green) and the aggregates were detected in FL2 channel (red), and the ratio of FL2/FL1 was calculated for determination of mitochondrial membrane potential.

Twenty thousand cells were seeded in 35 mm dish (Microtech) (70% confluence) and leave to adhere overnight. Mitochondrial morphology was assessed after cell staining with 10 nM Mitotracker CMX-Red (Invitrogen) for 30 min at 37°C. Fluorescence was visualized with a digital imaging system using an inverted microscope (Nikon, Japan). Images were captured with a Photometrics Cascade CCD camera system (Crisel) and analyzed with Metamorph acquisition/analysis software. The network morphology was assessed by Metamorph analysis: cells with shape factor ≥0.650 were classified as fragmented network, cells with shape factor <0.650 as tubular network.
Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining kit (Sigma, CS0390) according to manufacturer's instruction.

MMP was measured using a JC-1 staining kit (Sigma-Aldrich, Shanghai, China) according to the manufacturer’s instructions. Briefly, MEM-washed cells were stained with JC-1 dye for 20 min. Then, the cells were observed, and the data were collected with a fluorescence microscope and a multifunctional microplate reader.

Cultured iNeurons were grown in imaging dishes (Chamber slide Lab-Tek II 4; Fisher). To visualize mitochondria, the cells were transfected with a Mito-GFP plasmid two days before the analysis. To visualize the lysosomes in live cells, iNeurons were incubated with 250 nM Lysotracker-Red DND-99 dye (Invitrogen) in medium at 37°C for 30 min. After washing twice with PBS the medium was replaced with fresh medium. Images were taken one frame every 3 sec, for 3 min. Detection of mitochondrial membrane potential changes in live iNeurons were performed using membrane-permeant tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining kit (Sigma) according to manufacturer's instruction. To measure the speed of mitochondria, kymographs were generated and analyzed using ImageJ (Multiple Kymograph plugin). For psychosine toxicity test, cells were treated with psychosine and then imaged for 3 h, 2 frames every 30 min. Live imaging was acquired using a DeltaVision fluorescence microscopy system (Applied Precision) installed at the Hanyang Center for Research Facilities, Seoul.

Mitochondrial depolarization occurring in apoptosis is indicated by an increase in the green/red fluorescence intensity ratio following JC-1 dye staining. Detection of altered cell mitochondrial membrane potential was performed using a JC-1 staining kit (Sigma-Aldrich, St. Louis, MO). SCs were washed with PBS and stained with 2 µg/ml of JC-1 dye at 37 °C for 30 min in the dark. Photographs were taken using a fluorescence microscope (Olympus IX70, Tokyo, Japan) equipped with a digital camera (DP71, Olympus) at 520 nm excitation/596 nm emission. Fluorescence intensities were analyzed using ImageJ software (Rasband WS, National Institutes of Health).