70 mm cell strainer

PBMCs were thawed at 37°C and washed with cold medium [Roswell Park Memorial Institute (RPMI)-1640 with 10% fetal bovine serum] for subsequent experiments. Cells were resuspended in PBS and incubated with maglev magnetic beads (QDSphere, Beijing, China) to excluded dead cells. Afterward, cells were suspended in PBS with 0.04% BSA, passed through a 70-mm cell strainer (Thermo Scientific, Waltham, USA), and counted with Luna™ automated cell counter (Logos Biosystems, Anyang-si, South Korea). Cell counts were adjusted and loaded with 20,000 cells in the Chromium™ Controller for partitioning single cells into nanoliter-scale gel bead-in-emulsions (GEMs). Single Cell 3′ reagent kit v3 was used for reverse transcription, cDNA amplification, and library construction of the gene expression libraries following the detailed protocol provided by 10x Genomics (Chromium Single Cell 3′ Reagent Kits v3, Pleasanton, USA). Sequencing was performed using Hiseq XTen PE150 sequencer (Illumina, San Diego, USA).

Mice were humanely euthanized, and mouse tumors were harvested. Whole tumors were cut and minced into small pieces, followed by digestion in Liberase TL Research Grade 10 (2 µg/mL, Roche, 05401020001) and DNase I (Sigma, 260913-10 MU) in DMEM at 37 °C for 30 min. The digested tissue was filtered through a 70-mm cell strainer (Thermo Fisher Scientific). Cell suspensions were stimulated with PMA (100 µg/mL) plus the protein transport inhibitors Golgi stop and Golgi plug (1:1000, BD Bioscience, 51-2301 KZ) at 37 °C. After 4 h, live cells were identified by vivid yellow staining (Invitrogen, cat# L34959). Cells were stained for cell surface markers including CD45.2 (1:100, BioLegend, 109814), CD8 (1:100, BioLegend, cat# 100708), CD11c (1:100, BioLegend, 117311), and IA/IE (1:100, BioLegend, 107608) at 4 °C for 30 min. For intracellular staining, cells were fixed and permeabilized with a fixation/permeabilization kit (BD Bioscience, 554714) for 30 min at 4 °C and then stained with anti-IFN-γ (1:100, BioLegend, 505813) and anti-granzyme B (1:100, BioLegend, 515406) antibodies. Cells were imaged on a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar).

To isolate cells from the human aortic valve specimens, the tissues were first minced into pieces no larger than 1 mm³ and large calcified nodules are removed (Supplementary Video 2). Then tissues were transferred to a solution of 5 ml of digestion medium. This medium consisted of PBS, 0.5 mg/ml collagenase type I (from Sigma-Aldrich), and 0.5 mg/ml collagenase type II (from Sigma-Aldrich). The samples were then incubated at 37 °C for 30 min, with manual shaking occurring every 5 min. Following incubation, the samples were vortexed for 10 s and pipetted up and down for 1 min using a 5 ml pipette. Subsequently, 15 ml of ice-cold PBS containing 0.04% Bovine Serum Albumin (BSA, from Thermo Fisher Scientific) were added, and the samples were filtered through a 70 mm cell strainer (Thermo Fisher Scientific). The undigested calcification fragments were discarded, and the pelleted cells were washed with red blood cell lysis buffer (Miltenyi Biotec) to eliminate any residual red blood cells. After washing with PBS containing 0.04% BSA, the cells were resuspended in the same solution and filtered once more through a 40 μm cell strainer. The dissociated single cells were then stained with Calcein-AM (from Thermo Fisher Scientific) to assess viability, and further enriched with a MACS Dead Cell Removal Kit (Miltenyi Biotec).