Poly d lysine coated 8 chamber polystyrene vessel tissue culture treated glass slide

BV2 murine microglial cell line or purified primary microglia by FACS were plated at a density of 5 × 10⁴ and 2 × 10⁵ cells, respectively, on poly-D-lysine-coated 8 chamber polystyrene vessel tissue culture treated glass slide (BD Falcon). Cells were left to adhere overnight in DMEM at 37 °C under 20% O₂ with 5% CO₂ followed by stimulation with 100 ng/mL LPS (Calbiochem), 50 ng/mL IFN-γ (Peprotech), 20 ng/mL IL-4 (Peprotech), or 150 μM CoCl₂ (Sigma) for 6 hr. 3 μL of 1:10 diluted (1.1 × 10⁹ beads) 1 μm carboxylate-modified fluorescent microspheres (Invitrogen) were added and incubated for another 2 hr at 37 °C under 20% O₂ with 5% CO₂. Following incubation, medium was removed and ice-cold PBS was added to arrest the bead uptake. Cells were then fixed with 4% PFA, chambers were removed, and slides were mounted with Prolong Gold antifade reagent and analyzed by fluorescence microscopy as described above. The number of beads ingested per cell were counted for 100 cells from at least three independent experiments.

DCF-DA (2’,7’-dichlorodihydrofluorescein diacetate; Molecular Probes) was used to study the intracellular ROS production in BV2 cells and primary microglia. The study was performed on poly-D-lysine-coated 8 chamber polystyrene vessel tissue culture treated glass slide (BD Falcon), wherein the cells were seeded at a density of 1 × 10⁵ cells in 500 μl of 10% FBS supplemented medium at 37 °C under 20% O₂ with 5% CO₂. After 4 hr of cell attachment, DCF-DA was added at a concentration of 10 μM in fresh media 500 μl/well and incubated for 30 min. Following incubation, the dye solution was removed and the cells were washed twice with 500 μl/well in PBS. DCF-fluorescence was determined by fluorescence microscopy and flow cytometry.