Im35 microscope

Using Zeiss IM35 microscope (Zeiss) and a digital camera (Nikon Digital Sight DSL1), we determined the dimensions of the cells in control conditions after treatment with DMSO, MC2791, NAC and NAC plus MC2791. Dimensions are expressed in μm.

BCPAP cells were seeded into 12-well plates at a density of 1 × 10⁵ cells per well. After cells reached 100% confluency, the adherent cell layer was wounded by scraping two crossing perpendicular lines with a sterile 200 µL tip. After a wash with PBS to eliminate detached cells, fresh RPMI supplemented with 2% FBS was added to control cells while RPMI supplemented with 2% FBS and enriched with EVs isolated from CAL-62 was added to the remaining wells in order to evaluate the migration rate. Wounds were observed and photographed under a Zeiss IM35 microscope (Zeiss) and a digital camera (Nikon Digital Sight DS-L1) at 0, 24, 48, and 72 h after the scratch was made. The wound closure was the mean gap in µm of five randomly chosen fields and was measured using Nikon Digital Sight DS-L1 at 10× magnification.

For chemotaxis assays, transwell migration chambers were used according to the manufacturer’s protocols (Corning Incorporated, New York, NY, USA). Briefly a suspension of 10⁶ cells was added to the upper chamber of the transwells and allowed to settle for 2 h. The transwells were then moved to the wells containing GO-BSA (1.5 μg/mL or 3 μg/mL) and the substances used as positive or negative controls, i.e., LPS (L4391 Sigma–Aldrich) and necrotic extracts (10 ng/mL) obtained from freezing and thawing pellets of the anaplastic thyroid cell line CAL-62 (5 × 10⁶ cells). The transwell filters were then stained with 0.5% crystal violet (Merck; C0775) and analyzed using Zeiss IM35 microscope (Zeiss) and a digital camera (Nikon Digital Sight DSL1, Tokyo, Japan). Finally, according to the manufacturer’s instructions, stained cells were dissolved with 10% acetic acid (100 µL/well) for colorimetric reading of OD at 560 nm with Glomax^®-Multi Detection System (Promega, Milan, Italy) [29 (link)].