Confocal Microscopy, Live‐Cell Imaging, and Analysis

For live imaging, transfected cells were cultured in 35 mm glass‐bottom tissue–culture dishes (MatTek, Ashland, MA). The glass bottom was pre‐coated with collagen type I solution, 50 μg/ml (BD Bioscience, Tewksbury, MA) at 4°C overnight. Transfected cells were cultured for 36–40 h prior to imaging using confocal laser point scanning microscope (Leica, TCS SP5, Mannheim, Germany). To maintain 37°C, 5% CO_2, and 95% humidified atmosphere throughout imaging, the microscope was equipped with a temperature control cube (Life Imaging Services, Basel, Switzerland), with CO₂ and humidity control (OkoLab, Pozzuoli, NA, Italy). Time‐lapse imaging in four dimensions (X, Y, Z over time) was recorded with 12 bit camera at 400 HZ speed using a HC PL APO 63×/1.40 objective (Leica, Mannheim, Germany). GFP fluorescence was detected using an Argon Laser (488 nm, 20% output) with 8–10% laser power. Mark and Find function was used to record multiple cell positions every half hour, and the z‐slice thickness was maintained at ∼0.5 μm. During imaging, cells were treated with DMEM media with or without IL‐1α (10 ng/ml, GenScript, NJ) for 2 h prior to administration of PEMF for 4 h (IL‐1α was present together with PEMF during the whole experiment).

Imaris software (Bitplane, MA) was used to analyze the images captured on the confocal microscope. With the tracking module of Imaris, the GFP particles within a 3D volume of the cells were counted, tracked, and analyzed according to the threshold of particle size (0.5 μm) and of intensity.