In vitro stimulation of microglia
CdA powder was provided by Merck. CdA was dissolved in dimethyl
sulfoxide (DMSO) (Sigma-Aldrich, D2438). Microglia were treated with
0.2% DMSO, LPS (10 ng/ml) (Sigma-Aldrich, L2630), DMSO and LPS, one of
four concentrations of CdA (0.01 µM, 0.1 µM, 1 µM and 10 µM) alone or in
combination with LPS for 24 hours. For migration, the cells were
stimulated immediately before placement in the IncuCyte. Microglia were
stimulated with IL-4 (20 ng/ml) or LPS (10 ng/ml) alone or together with
CdA for 24 hours for qPCR and Meso Scale.
MTT viability
assay
MTT solution (0.5 mg/ml) (Sigma-Aldrich, M0283) was added to LPS- and
CdA-pretreated microglia for 4 hours. The absorbance was measured by a
microplate reader (Molecular Devices, 6465).
Phagocytosis assay,
size and granularity
Microglia were incubated with fluorescent latex beads and analyzed by
flow cytometry as described (20). Fluorescent latex beads (Polysciences,
Inc., 17154-10) were added to LPS and CdA treated microglia for 40 min
at 37°C (sample) and 4°C (control). Cytochalasin D (5 µg/ml) was applied
prior to addition of the beads as negative control. Phagocytosis was
stopped by placing the cells on ice. Cells were detached by using 0.2%
Trypsin-EDTA and analyzed by flow cytometry. Microglia size and
granularity were assessed by flow cytometry by measuring forward scatter
(FSC) and side scatter (SSC), respectively.