Cd16.2 fcγriv

To study macrophage polarization, leukocytes were isolated from freshly harvested brain tissue following previously published protocol [83 ]. The protocol was slightly modified and is included in the supplementary information. As previously, Fc receptors were blocked with antibodies against CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE-Cy7 CD45 (BioLegend, USA; clone, I3/2.3), PE F4/80 (BioLegend, USA; clone, BM8), Alexa Fluor 488 CD86 (BioLegend, USA; clone, GL-1), and Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2). Macrophages were defined as CD45^high, F4/80⁺. Cells were run in a Novocyte 2060 (ACEA Biosciences, USA) at a rate of 14 μL/min.

For MSC cell-surface characterization, the following antibodies were used: APC-Vio770 CD29 (Miltenyi Biotec; clone, HMβ1-1), VioBright FITC CD44 (Miltenyi Biotec, USA; clone, REA665), PerCP Sca-1 (Biolegend, USA; clone, D7), PE CD31 (Miltenyi Biotec, USA; clone, 390), PE CD45 (Biolegend, USA; clone, 30-F11), and PE-Vio770 CD105 (Miltenyi Biotec, USA; clone, MJ7/19). For analysis of in vitro macrophages, we first blocked Fc receptors with CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE CD45 (Biolegend, USA; clone, 30-F11), Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2), and PE CD163 (ThermoFisher, USA; clone, TNKUPJ). A Novocyte 2060 (ACEA Biosciences, USA) was used to measure mean fluorescence intensity of GFP from transfected MSCs, MSC cell-surface markers, and macrophage markers. Data was analyzed in FlowJo software (BD Biosciences, USA).