Spinning disk confocal head

PaTu-S.iPLK4 cells untreated or induced to have amplified centrosomes (48 hours 2 μg/ml DOX treatment) were cultured for 48 hours in vesicle depleted media before the conditioned media was collected. EVs were then harvested from the conditioned media by ultracentrifugation alone, or in combination with SEC as described previously. EV number was then quantified by ImageStream as described above. 20 million EVs were then added to the culture medium of PS1 cells that had been plated on glass coverslips at a density of 1x10⁴ cells 24 hours prior. 48 hours later, a second dose of 20 million EVs was administered. 24 hours later cells were fixed and stained for αSMA and DNA as described previously. Images were acquired using an inverted Nikon microscope coupled with a spinning disk confocal head (Andor) with a 40x objective. PS1 activation was quantified based on α-SMA organization, where the formation of α-SMA fibers was used as a measure of activation. Resting PS1 cells display diffuse αSMA staining consistent with low to no activation. Increase expression of αSMA with the appearance of αSMA fibers is indicative of PS1 activation and we as classify strong activation PS1 cells where the majority of αSMA is associated with fibers displaying increased intensity levels. Roughly 150 cells were quantified manually per condition. All conditions were quantified blindly.

Collagen gels were performed as previously described (Infante et al, 2018 (link)). Briefly, glass coverslips were layered with 15 μl of a 2.2 mg/ml type‐I collagen solution (bottom layer). Polymerization was induced at 37°C for 3 min. Then, a cell suspension (1.5–2.5 × 10⁵ cells/ml) was added to the bottom layer and cultures were incubated for 30 min at 37°C to allow cells to adhere to the collagen gels. Growth medium was gently removed and a 2.2 mg/ml type‐I collagen solution was polymerized on top of the cells (top layer). After polymerization at 37°C for 90 min, growth medium was added to the cultures. Z‐stacks of images were acquired with an inverted Nikon microscope coupled with a spinning disk confocal head (Andor) with a 60× objective.

The Magic Red Cathepsin B kit (Bio-Rad, ICT937) was used to analyze the protease activity of Cathepsin B in lysosomes as a proxy to lysosome function. In the presence of functional cathepsin B, the Magic Red substrate is cleaved allowing the Cresyl violet fluorophore to fluoresce red upon excitation at 550-590 nm. Briefly, cells to be analyzed were plated on coverslips and the Magic Red substrate (Magic Red stock was reconstituted in 50 μl DMSO and diluted 1:10 in deionized water) was added to the growth media (20μl was added per 300μl of growth media as per the manufacturer’s instructions) for the final hour of the experiment. Cells were then fixed in 4% formaldehyde as previously described. Cresyl Violet fluorescence was detected using an inverted Nikon microscope coupled with a spinning disk confocal head (Andor). Z stack images were acquired, and sum intensity image projections were generated using ImageJ. Fluorescence intensity was then quantified per cell with ImageJ⁶⁶ (link). All conditions were quantified blindly.