Chamber slide lab tek 2 4

For live cell imaging, Jurkat cells were grown in imaging dishes (Chamber slide Lab-Tek II 4; Fisher). The cells were transfected 18 hrs before the experiment. The cells were loaded with 1 μM ER Tracker Blue-White DPX or LysoTracker Blue-DND-22 (Invitrogen/Molecular Probes) to label the ER or lysosomes, respectively. Surface staining of wildtype or mutant EGFP-tagged CLEC4C-expressing cells was performed 15 min prior to imaging. Pure anti-CD303 (clone AC144, mouse IgG1, Miltenyi Biotec) was applied to transfected live Jurkat cells to detect the surface expression of CLEC4C in both wildtype and mutant constructs. Ten minutes after the addition of the primary antibody, anti-mouse secondary TRITC was added for 5 min and the cells were monitored. The cells were imaged live in growing medium using a DeltaVision fluorescent microscopy system (Applied Precision). Images were taken for 15 min, 1 frame every 1 min.

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.