Axio observer microscope

Geltrex LDEV-free reduced growth factor basement membrane matrix (Gibco, Burlington, ON, Canada, cat# A1413201) was used. The Geltrex basement membrane matrix was thawed overnight in a 4 °C fridge. Once thawed, it was mixed well and aliquoted (80 μL/well) into a 96-well plate (on ice) with chilled pipette tips. To prevent air bubble formation, the basement membrane was dispensed without a full stop, and the plate was centrifuged at 300× g for 10 min at 4 °C. Then, the coated plate was incubated at 37 °C and 5% CO₂ for 30 min. HUVECs were cultured and transfected as described above. The cells were serum-starved for 4 h and detached with trypsin-EDTA, then resuspended at 2 × 10⁵ cells/mL in complete EGM-2 medium. Each coated well received 100 μL of cell suspension (2 × 10⁴ cells/well). Tube formation was carried out at 37 °C under an atmosphere of 5% CO₂ and live-imaged with a Hamamatsu Camera and a Zeiss Axio Observer microscope (5× magnification) with Zen Pro 3.6 software. The images were processed at the 16 h time point with Zen 3.3 Lite (Blue Edition) and analyzed with Angiogenesis Analyzer 1.0 software for ImageJ2 or Fiji. All conditions were done in duplicate.

MCF-7 LTC or MDA-MB-231 LTC cells were seeded at a density of 3000 cells/well in 8-well LabTek glass-bottom chambers (#155409, Thermo Fisher Scientific, USA). The following day, live-cell imaging extending up to 24 h was initiated on a ZEN AxioObserver microscope equipped with a heated and humidified stage. Time-lapse images were acquired every 1 h using a 10x objective and Rolera camera. Post-acquisition analysis of single-cell tracks was performed manually on cells that did not undergo cell division using the ImageJ program. Speed was calculated by dividing track length at each time point divided by elapsed time (hours). Rose plots and persistence were computed using the MATLAB program. Persistence was calculated by dividing displacement with track length. Confined and nonconfined classification was determined from individual track mean square displacement curve fit alpha, with alpha values <1 corresponding to confined motion. From three independent experiments, at least 57 and 60 tracks were analyzed in MCF-7 LTC and MDA-MB-231 LTC cells, respectively.

To assess the effect of pEVs on EV recipient cells, we used endothelial cells, because endothelial cells are the only resident cells in the LAA that are in direct contact with blood-derived EVs. As endocardial endothelial cells from the LAA are not commercially available, we used female primary human coronary artery endothelial cells (HCAECs) from passage 8 (Promocell, Cat# C-1222) for basic migration experiments in a scratch-wound assay. The assay was performed as previously described [39 (link)]. In brief, HCAECs were allowed to grow to confluency in a 6-well plate. The pEVs from 500 µl of supernatant were resuspended in 500 µl fresh RPMI medium and further diluted 1:1 in Endothelial Cell Growth Medium MV with supplements (Promocell, Cat# C-22020) before incubation with the HCAECs. Directly after stimulation, a scratch was applied to the center of the well with a sterile 200 µl pipet tip. The scratch was photographed in a marked position at 0, 2, 4, and 6 h. To calculate the migratory activity of the cells, the remaining cell-free area was measured and compared to the cell-free area at 0 h. Image acquisition and analyses were conducted with a Zeiss Axio Observer microscope and the ZEN 2.3 pro software.