. Briefly, cells were seeded in a 15 cm at a density appropriate for them to reach confluency after 24 h. Samples were processed separately to ensure rapid isolation of lysosomes using buffers that were pre-chilled on ice. Cells were quickly rinsed twice with ice-cold PBS buffer and then scraped in KPBS , supplemented with Protease Inhibitors, and collected by centrifugation at 1000 ×for 2 min at 4 °C.
For the quantification of the number of GAA and CI-M6PR particles, image were processed with the Imaris software. The ‘cell’ module was applied to identify nuclei, cell borders and spots: briefly, constant image smoothing and background subtraction were applied to identify nuclei, cells and particles, and kept constant along images. Moreover, nuclei were identified according to their threshold and diameter, and split by seed points.
To calculate the percentage of LC3-LAMP1+ autolysosomes, the number of LC3-GFP spots and LC3-LAMP1 overlapping area, confocal sections were acquired at the same laser power and photomultiplier gain. Images were then processed using the ImageJ software. Single channels from each image were converted into 8-bit grayscale images, and then thresholded in order to subtract background.
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