Phagocytosis

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Procedure

As an index of phagocytosis we used the uptake of 6 μm-diameter fluorescent latex
beads (Fluoresbrite Yellow green; Polysciences). The beads, purchased as aqueous
suspension of 2.5 % solids- latex (corresponding to 2x108 beads/ml), were opsonized
for 30 min at 37 °C in Kreb’s Ringer PBS containing 50 % of fetal calf serum and then
25 μL pipetted onto the slices, at a final concentration of 2x105 beads/25 μL. OHSCs
were incubated with the beads at 37 °C in 5 % CO2 for 4 hrs. In parallel, some OHSCs
were incubated with the beads at 4 °C to inhibit phagocytosis, and used as a negative
control. After incubation, OHSCs were washed twice with PBS for 2 min to remove
excess beads, and then fixed with 4 % PFA for 15 min at RT. After washing two times
with PBS, the attached non-ingested beads were lysated by a rapid (3 to 5 sec) rinse in
acetone. OHSCs were subsequently processed for Iba-1 staining as detailed above. The
latex beads were visualized using fluorescence microscopy and ingestion by Iba-1
positive microglia was confirmed by confocal analysis. A blind observer counted the
numbers of ingested beads in 10 randomly chosen fields (corresponding to 0.8 mm2
each)/slice/condition and the experiments were run in triplicate. The results are given as
the mean ± S.E.M. of the numbers of ingested beads/mm2.

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