To assess ROS generation in primary microglia, cell homogenates from 3- and 20-month-old male wild type mice and from 9-month old male GRN−/− mice were prepared and antibody staining was performed as described above (see Microglia Isolation ). Cell homogenates were incubated in FACS buffer with CellROX™ Deep Red (1:500, Invitrogen) for 30 min at 37°C, washed twice in FACS buffer, and CellROX™ Deep Red Intensity was analyzed on ARIA 3.1 (BD Biosciences).
To measure ROS in BV2 cells, cells were split into 24 well plates at 5×104 cells per well in DMEM +5% FBS and treated with LPS, Triacsin C and vehicle solutions for 18 hours. Next, cells were incubated in DMEM+5%FBS with CellROX™ Orange (1:500, Invitrogen) for 30 min at 37°C, washed twice in PBS, and CellROX™ Orange Intensity was examined by fluorescence microscopy (Keyence Corp., Osaka, Japan). Following microscopic analysis, cells were detached using TripLE, transferred to FACS tubes and CellROX™ Orange intensity was analyzed on BD AccuriC6 flow cytometer.
To measure ROS in BV2 cells, cells were split into 24 well plates at 5×104 cells per well in DMEM +5% FBS and treated with LPS, Triacsin C and vehicle solutions for 18 hours. Next, cells were incubated in DMEM+5%FBS with CellROX™ Orange (1:500, Invitrogen) for 30 min at 37°C, washed twice in PBS, and CellROX™ Orange Intensity was examined by fluorescence microscopy (Keyence Corp., Osaka, Japan). Following microscopic analysis, cells were detached using TripLE, transferred to FACS tubes and CellROX™ Orange intensity was analyzed on BD AccuriC6 flow cytometer.