Sa β gal

Cells collected from secondary lymph organs or CSF of patients were stained with fluorescent-conjugated antibody: fixable viability dye (Thermo Fisher Scientific, Cat# 65-0866-14), CD45 (BioLegend, Cat# 103132), CD3 (BioLegend, Cat# 100213), CD8 (Thermo Fisher Scientific, Cat# 53-0081-82, 17-0088-42), CD4 (Thermo Fisher Scientific, Cat# 62-00420-80), CD11b (BioLegend, Cat# 101222), Ly6G (BioLegend, Cat# 127613), Ly6C (BioLegend, Cat# 128017), CD11c (BioLegend, Cat# 117322), H-2Kb bound to SIINFEKL (BioLegend, Cat# 141605), OVA pentamer (ProImmune, Cat# F93-2A-G), SA-β-gal (Dojindo, Cat# SG03), and CD49d (Thermo Fisher Scientific, Cat# 12-0492-81, 16-0492-85) for 30 min at 4℃; primary antibodies: luciferase (Abcam, Cat# ab185924, 1:100), cleaved caspase-3 (Cell Signaling Technology, Cat# 9661, 1:200), and Ki-67 (Thermo Fisher Scientific, Cat# 14-5698-80, 1:50). For intracellular staining, cells were pretreated with Foxp3/Transcription Factor Fixation/Permeabilization kit (eBioscience, Cat# 00-5521-00) according to the manufacturer’s instructions. Flow cytometry was performed on Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software.

Senescence-associated β-galactosidase (SA-β-gal) staining was performed according to the instructions provided by the manufacturer (DojindoMolecular Technologies, Inc., Kumamoto, Japan). First, OCT-embedded liver tissues were cryosected at a thickness of 10 μm using a Leica CM3050S cryosectioner. Then sections were fixed in 4% paraformaldehyde for 20 min at RT and washed in PBS three times. SPiDER-βGal staining working solution was diluted to 20 μmol/L (Dojindo Molecular Technologies, SG03, 1:2,000) with McIlvaine buffer (pH 6.0) and sections were incubated with this working solution overnight at 4°C. Sections were then counterstained with Hoechst33342 (Thermo Fisher, H3570, 1:1000) at RT, washed with PBS three times, and mounted in VECTERSHIELD^® Anti-Fade Mounting Medium (Neobioscience, H-1000). Image acquisition was performed using a Zeiss LSM900 confocal system, and data analysis of the percentage of SPiDER-βGal positive cells was performed using image J.