Cd45 bv711

Cell suspensions of spleen and peritoneal cells were stained with fluorescent antibodies (Abs) for 10 min at 4°C for flow cytometry analyses. Analysis of 200,000 events gated on viable cells was performed on a BD LSRFortessa Cell Analyzer (BD Biosciences, NJ, USA). Results were analyzed with BD FACSDiva Software 6.0 (BD Biosciences). Fluorescent Abs (TCRβ-Biot, SAV-BV605, CD4-PB, NK1.1-PerCPCy5.5, CD19-FITC, CD45-APC, CD45-BV711, CD11c-PECy7, Ly6C-BV421 and Ly6G-APC) were obtained from BD and CD8-APCvio770, CD11b-FITC and F4-80-PE were obtained from Milteyni.

The mice used for flow cytometry analysis were euthanized and perfused with ice-cold PBS. The brains were removed and immediately placed into ice-cold HBSS. Brain samples were then dissociated using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). Dissociated cells were resuspended in 10 ml of 30 % Percoll solution (Sigma) in an RPMI medium and laid over a 1 ml 70 % Percoll solution layer. After centrifugation at 800g for 30 min at 4 °C, interphase cells were transferred to a new 15-ml Falcon tube and washed with RPMI. Cell pellets were resuspended with FACS buffer (DPBS with 0.5 % BSA fraction V) and blocked with one volume of blocking solution (5 % normal mouse serum, 5 % normal rat serum, 5 % normal rabbit serum, 2 % FBS, and 1 % BSA fraction V in ×1 DPBS) for 30 min and stained for 30 min with fluorophore-conjugated antibodies on ice (CD45-BV711, CD11b-AF700, Ly6C-Pacific Blue, and Ly6G-PE were purchased from BD Pharmingen); 7AAD was used to exclude dead cells. Data were collected on an Aria III sorter (BD) and analyzed with FlowJo v10 software (Tree Star Inc.). At least 20,000 and 200,000 viable events were collected from each brain and blood sample, respectively.