Cd8 fitc 53 6.7

BMNC. PECs, lymph or splenic cells were washed in FACS buffer, blocked for 10 min in F_c block and incubated with various conjugated antibodies. To determine antigen specific T cells: CD3-FITC, OTI-CD8-dsRed, MHC I-Ova dextramer-APC (Immudex, Denmark and Tetramer core facility NIH, Bethesda, MD); to determine T cell activation: CD3-PerCp (145-2C11), CD4-PECy7(RM4-5), CD8-FITC (53-6.7), CD44-PE (IM-7), CD62L-APC (MEL-14); to determine monocyte populations: CD11c-APCCy7 (N418), CD11b-FITC (M1/70), Ly6C-PE (AL-21), Ly6G-APC (RB6-8C5), MHCII-PECy7 (M5/114.15.2), and CD45-PerCp (30F11), (eBioscience, San Diego, CA). Cells were analyzed using a BD FACSCanto II flowcytometer (BD Biosciences, San Jose, CA) and FlowJo analysis software v.8.7.3 (Treestar Software, Ashland, OR). Cell populations were determined by gating on CD4⁺, CD8⁺, (T cells) CD45^hi/CD11b⁺ (macrophages) and CD45^int/CD11b⁺ (microglia) Ly6CintLy6G⁺, (neutrophils) Ly6C⁺Ly6G⁻, (inflammatory monocytes) from a live cell gate (gating strategy as depicted in Supplementary Figs. 5, 7).

Surface staining was performed with antibodies diluted in FACS-buffer (PBS, 2% FCS, 2 mM NaN₃, and 2 mM EDTA) for 20 min at 4 °C, followed by three washing steps. Blood samples were incubated with 2 ml FACS lysing solution (BD Bioscience. # 349202) to lyse erythrocytes. The antibodies used were CD11b-PE-Cy7 (M1/70, #25-0112-82), CD19-FITC (eBio1D3, #11-0193-82), CD8-FITC (53-6.7, #MA1-10303), CD4-PE (GK1.5, #12-0041-85), F4/80-PE (BM8, #12-4801-82), NK1.1-APC (PK136, #17-5941-82) from eBiosciences or IFN-γ-FITC (XMG1.2, #562019) from BD Pharmingen. All antibodies were used at a 1:150 dilution in FACS buffer. For proteostat® staining, the organs were collected in 1× PBS and a single cell suspension was prepared by digestion with 1 mg/ml DNAse I (Sigma, #DN25) and 1 mg/ml collagenase D (Roche, #50-100-3282) in HBSS (10 mM Hepes) in a gentleMacs Dissociator (Miltenyi Biotec). Proteostat® staining was performed using an Aggresome detection kit (Enzo®, #ENZ-51035-K100) according to the manufacturer’s protocol. Samples were measured using FacsVerse (BD Biosciences). The gating strategy for flow cytometry is depicted in Supplementary Fig. 2. Flow cytometry data were analyzed with FlowJo v10 (BD Biosciences).