Mouse, pig, and human atherosclerotic lesions have common and distinct mesenchymal cell populations
Mouse, pig, and human atherosclerotic lesions have common and distinct mesenchymal cell populations
Animals were euthanized, and the right coronary arteries were quickly isolated and opened. The atherosclerotic plaques were dissected without media. Approximately 200-250 mg of plaque was selected, washed in HBSS, and minced. It was incubated for 60 minutes at 37 °C in 1 ml of an enzyme solution containing 1,5 mg/ml (8 U/ml) of elastase (LS002279, Worthington Biochemical), 2 mg/ml of liberase (05401119001, Sigma Aldrich) and 300 µg/ml of DNaseI (Sigma Aldrich, DN25). Non-digested tissue was disaggregated using a smooth-tipped Pasteur pipette. Reactions were stopped by adding PBS containing 0.5% bovine serum albumin (BSA). The suspension was passed through a 70 µm pore filter to remove aggregates. Cells were collected by centrifugation and resuspended in 500 µl PBS+0.5% BSA. DAPI (final concentration, 1 µg/ml final ) and Draq5 (Thermo Scientific 65-0880-96; final concentration, 5 µM) were added to enable detection of viable and dead cells. Viable cells (Draq5 positive and DAPI negative) were sorted with a FACSAria Cell Sorter. Atherosclerosis was induced in PCSK9D374Y transgenic minipigs by feeding a standard pig feed supplemented with 20% w/w lard, 1.5% w/w cholesterol (HFHC), 0.7% NaCl w/w, from 3 until 18 months of age. Up to 16500 cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics). After reverse transcription and cDNA amplification, sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).