Cryostor cell cryopreservation medium

Peripheral venous blood was collected in K2-EDTA blood tubes (BD Vacutainer). For scTCR-seq, blood was processed within 10 minutes after withdrawal (cohort 2). For both cohort 1 and cohort 2, blood was diluted 1:2 in PBS containing 2% FCS. A density gradient was created using SepMate PBMC isolation tubes (STEMCELL Technologies) containing Ficoll-Paque Premium (GE Healthcare). Cells were centrifuged at 1,200g for 10 minutes at room temperature. The intermediate layer containing PBMCs was isolated and washed twice with PBS + 2% FCS (250g, 10 minutes, room temperature). Cells were taken up in PBS + 1% BSA until further processing. For cohort 3, whole blood samples were lysed twice with ACK lysis buffer in PBS (1:10) for 10 minutes at room temperature and washed with PBS (300g, 5 minutes). Cells were taken up in RPMI + 1% FCS and cryostored in CryoStor cell cryopreservation medium (Sigma-Aldrich) until further use.

Human carotid plaques were collected during CEA; the culprit segment (5 mm) was used for histology and embedded in paraffin as described elsewhere^{48 (link)}. In brief, culprit segments were fixed in 4% formaldehyde and decalcified in 10% EDTA, pH 7.5. Afterwards, culprit segments were embedded in paraffin. Time between surgical removal and plaque processing did not exceed 10 minutes. The inclusion of a small medial layer in the dissected tissue could not be excluded during the surgical procedure. The remainder of the plaque was washed in RPMI and minced into small pieces with a razor blade. The tissue was then digested in RPMI 1640 containing 2.5 mg ml⁻¹ of collagenase IV (Thermo Fisher Scientific), 0.25 mg ml⁻¹ of DNAse I (Sigma-Aldrich) and 2.5 mg ml⁻¹ of Human Albumin Fraction V (MP Biomedicals) at 37 °C for 30 minutes. In cohort 2, 1 µM flavopiridol (Selleck Chemicals) was added to the digestion mixture. Subsequently, the plaque cell suspension was filtered through a 70-µm cell strainer and washed with RPMI 1640. Cells were kept in RPMI 1640 with 1% FCS until subsequent staining for flow cytometry (cohort 1), feature barcoding and FACS (cohort 2) or cryostored in CryoStor cell cryopreservation medium (Sigma-Aldrich) until further use.