Model axiovert 100 inverted microscope

Scratch assay was performed using silicone Ibidi inserts. Inserts were place in 12-well plate and 70 µL of standard BV2 medium with 35,000 cells were seeded in each compartment. After 24 h, inserts were removed, and the wells were filled with serum-free medium with or without LPS (100 ng/mL) [20 (link)] or ATP (300 µM) [71 (link)]. Cells were sham-treated or stimulated with ELF-EMS (20 min of 13.5 mT/60 Hz) 30 min after seeding. They were allowed to migrate for 24 h. Four pictures per sample were taken (20× magnification) 24 h after ELF-EMS with a Zeiss Model AxioVert 100 Inverted Microscope. The area occupied by migrated cells was quantified using Image J (version 1.53i) software [72 (link)]. Results are presented normalized to control culture.

Cell transmigration assays were performed using Corning Transwell membrane filters (8 µm pore size, Corning Costar, New York, NY, USA). Inserts on both sides were coated with fibronectin (10 µg/mL). BV2 cells were placed overnight on serum-free medium, and the next day seeded at a density of 104 cells/insert in 100 µL serum-free medium. Bottom wells were filled with serum-free medium (0%) (Control), with LPS (100 ng/mL), or ATP (300 µM) as chemoattractant. BV2 cells were allowed to migrate for 6 h. Primary microglia were seeded at a density of 10⁴ cells/insert and were allowed to migrate for 24 h. Bottom wells were filled with 10:10:1 complete medium with or without ATP (300 µM) [71 (link)]. Cells were sham-treated or stimulated with one session of ELF-EMS (20 min of 13.5 mT/60 Hz) 30 min after seeding. Once migration time was completed, cells were fixed for 5 min in 4% PFA, washed with PBS, and stained for 2 min with 0.05% crystal violet. Migrated cells were at the bottom side of the insert, thus the upper side of the inserts were swapped with cotton buds. From each insert, four pictures of the bottom side were taken on 10× magnification with a Zeiss Model AxioVert 100 Inverted Microscope. The area occupied by migrated cells was quantified using Image J software. The results are presented normalized to control culture (no ATP, no EMF treatment).