Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

William Binning; Aja E. Hogan-Cann; Diana Yae Sakae; Matthew Maksoud; Valeriy Ostapchenko; Mohammed Al-Onaizi; Sara Matovic; Wei-Yang Lu; Marco A. M. Prado; Wataru Inoue; Vania F. Prado

Ca2+ imaging was performed as described previously (Beraldo et al., 2016) with some modifications. Cultures were washed with Krebs-Ringer HEPES (KRH) buffer (129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 2.8 mM glucose, 10 mM HEPES) supplied with 1 mM CaCl2, incubated for 45 min with 5 µM of red spectrum fluorescent dye Rhod-2 AM at 37 °C, washed again 3 times with calcium-free KRH and incubated in KRH with or without calcium for 10 min at 37 °C. Intracellular Ca2+ was recorded with a FV1000 confocal microscope equipped with a 30x/1.05 objective. The imaging field was chosen to contain cells expressing hM3Dq as shown by mCitrine fluorescence (ex488/em505-550). Time kinetics were done with a 2.5-second step. After 100 s of background recording, 3 µM CNO, 1 mM ATP and 2 µM ionomycin or 10 µM thapsigargin (for KRH with or without calcium, respectively) were added consecutively to the dish. Rhod-2 fluorescence was registered using 546 nm excitation laser and 560-660 nm emission filter. ROIs were assigned and quantified for each cell in the imaging field using ImageJ (NIH, Bethesda, MD). Integrated fluorescence was normalized to the average signal during background recording. At least three independent cultures were obtained from three different litters and at least 50 cells were imaged for each condition.