After 48 hours of stimulation, primary astrocytes were detached and washed once with cold 1× PBS. Live/dead staining was performed with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, L34957) according to the manufacturer’s instructions. Surface staining was performed at 4°C in the dark for 20 minutes with Abs diluted in FACS buffer (1× PBS, 2% FBS, 2 mM EDTA). Cells were then washed twice with FACS buffer subsequently fixed for intracellular staining using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience, 00552300) according to the manufacturer’s instructions. The following Abs were used: BV421-CD11b (Biolegend, 101235), PE-Cy5.5-CD45 (Thermo Fisher Scientific, 35045180), PE-eFlour610-iNOS (eBioscience, 61592080), PerCP-eFlour710-TNF (eBioscience, 46732180), APC-GM-CSF (eBioscience, 17733182), and Ki67-AF700 (Biolegend, 652419).
Cells were acquired on a 3L Cytek Northern Lights flow cytometer. Analysis of flow cytometry data was performed with the OMIQ platform.
Cells were acquired on a 3L Cytek Northern Lights flow cytometer. Analysis of flow cytometry data was performed with the OMIQ platform.