Ca2+ Imaging of Microglia Activation

Mouse macrophages and microglia were washed with HBSS and then loaded with 2 µM Fluo8‐AM solution (Stratech) at room temperature in the dark for 1 h. Cells were then washed and imaged in Ca²⁺‐free media and EGTA used as a negative control. Plates were warmed in a plate reader (Spectramax Gemini EM) or automated cell screening system (FLIPR Penta Molecular Devices) to 37°C and cells were exposed at set time points to anti‐FcγRII/III, LPS, oligomers of Aβ1–42 or DOPS‐liposomes (25 μg/ml), as above. This was followed by ionomycin (2 µM) as a positive control. Levels of fluorescence were detected at ex 490 nm/ em 520 nm (Hagen et al, 2012 (link)). Cells were inhibited by pre‐exposure for 2 h with edelfosine (10 µM) (Tocris) or U73122 (5 µM) (Sigma) or xestospongin C (5 µM) (Abcam). Similarly, hiPSC‐derived microglia were exposed to anti‐CD32 (Fisher 16‐0329‐81, 5 μg/ml) or DOPS‐liposomes (25 μg/ml).