Aggresome detection kit

MCEC cultures in 12-well format were trypsinized, washed and fixed using 4% Paraformaldehyde. Following permeabilization, cells were stained for aggresomes using the Aggresome detection kit (#ENZ51035, Enzo Life Sciences, NY) following manufacturer’s instructions. The aggresome detection reagent emits strong fluorescence when bound by misfolded proteins but not in solution. Therefore, the increase in fluorescence intensity is indicative of the levels of protein aggregates present in the cells. After filtration using 50 μm sterile CellTrics Filters flow cytometry analysis was conducted on MACSQuant VYB. 10,000 cells were collected per acquisition. Cells treated with ER stress inducer, Thapsigargin (#1138, Tocris, Minneapolis, MN, United States), served as a positive control, unstained cells were used as negative control. Data were analyzed with FCS Express.

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).