Cellsens entry software version 1.5

For microscopy, cells were plated at 50,000 cells/chamber on Nunc Lab-Tek II 8-chamber slides (Fisher Scientific, Pittsburgh, PA, USA) and treated in the same manner as the 96-well plates. The medium was aspirated, the chambers washed with Dulbecco’s PBS (DPBS, Gibco), and cells were fixed for 5 min with ice-cold methanol. After removing the methanol, the slides were air-dried and then stained with Giemsa (Gibco) stain (1:20 dilution of stock) for 10 min followed by extensive washing with tap water. Images were taken with an Olympus BX61 Microscope equipped with a D72 camera and processed with cellSens Entry Software (version 1.5) (Olympus America, Melville, NY, USA).

Cells were plated onto 96-well plates at 5000 cells/well (astrocytes) or 2500 cells/well (BMEC) 100 μL per well, and allowed to incubate at 37 °C, 5% CO₂ for 24 h. Media was then removed and cells treated with media spiked with metal or organic compounds in a serial dose dilution ranging from 0 μM to 1000 μM and incubated for an additional 24 h prior to viability assays. For images, cells were plated at 50,000 cells/chamber on Nunc Lab-Tek II 8-chamber slides, treated in the same manner as the plates. Medium was aspirated, the chambers washed with Dulbecco’s PBS (DPBS, Gibco) and cells fixed with ice-cold methanol for 5 min. After removing the methanol, the slides were air dried before being stained with Giemsa (Gibco) stain (1:20 dilution of stock) for 10 min followed by extensive washing with tap water. Images were taken with an Olympus BX61 Microscope with D72 camera and cellSens Entry Software (version 1.5) (Olympus America, Melville, NY, USA).

Liver tissue, sWAT, eWAT, and BAT were fixed in 4% PBS-buffered paraformaldehyde, dehydrated using an ascending ethanol series, and embedded in paraffin. Sections (4 μm) were prepared and stained with hematoxylin/eosin (HE) to visualize tissue structures. Representative photos were taken using Microscope Axio Imager M1, (Carl Zeiss Jena, Jena, Germany) and CellSens Entry Software Version 1.5 (OLYMPUS Cooperation, Shinjuku, Japan).