Podonut (podosome) formation requires Ca
entry, likely through Orai1/CRAC channels. (A-F). Microglia were fixed and stained for F-actin (phalloidin; green), talin (red), and the nuclear stain, DAPI (blue) after 30 min (37°C) incubation in: standard bath (control, (A)), Ca2+-free bath solution containing EGTA (B), 5 μM gadolinium (Gd3+, (C)), 50 μM 2-APB (D), 10 μM BTP2 (E) or 100 μM spermine (F). Scale bars, 20 μm. G. Percentage of microglia expressing podonuts under each condition. Cells were fixed after 30 min treatment. Podonuts were counted from three random fields on each immunostained cover slip, and averaged for cultures prepared from the number of rats indicated on each bar. Results are expressed as mean ± SEM, and ***P <0.001 indicates differences from cells in standard bath (control).