Latex bead solution

BV-2 cells were pre-treated with DMSO as vehicle, 50 μM I3C, vehicle +50 ng/ml LPS, and 50 ng/ml LPS + 50 μM I3C for 4 h. After treatments, 2 μl latex bead solution (polystyrene microparticles, Sigma Aldrich; St. Louis, MO, USA) was added to the wells for 4 h to determine the influence of I3C on phagocytosis. Five micrographs per well were taken using an AxioVert.A1 inverted microscope (Carl Zeiss; Germany). The phagocytic activity was determined by calculating the number of cells, which phagocytosed 10 or more latex beads.

Carboxylate-modified red fluorescent latex beads (2 μm diameter; 4 μl of 2.5 % latex bead solution; Sigma-Aldrich) were opsonized in 10 % FBS with DMEM or DMEM/DF12 (6 ml) for 60 min at 37 °C. Subsequently, cells were washed twice with ice-cold PBS to remove uningested particles, and fixed with 4 % paraformaldehyde. The fluorescence of macrophages or microglia were measured by flow cytometry. Results were presented as a percentage of the phagocytic cell population (number of cells that ingest at least 1 bead/total number of cells × 100 %) and phagocytosis index (average number of ingested beads per cell = number of ingested beads/total number of cells).