Mus musculus
Protocols: Aortic arch and root samples from Apoe-/- mice fed a Western diet for 6 weeks were collected after treatment with either rHDL or PS@rHDL (n = 6 per group). Mice were fasted for 24 hours and following euthanasia were perfused with 5 mM EDTA PBS (Sigma-Aldrich, E1644). After adipose tissue removal, aortic arch and root samples were diced and digested with a cocktail of enzymes, including liberase TH (4 U/mL; Sigma-Aldrich, 5401151001), hyaluronidase type I-S (60 U/mL; Sigma-Aldrich, H3506) and deoxyribonuclease I (40 U/mL; Sigma-Aldrich, DN25) in PBS at 37ºC for 1 hour in an Eppendorf ThermoMixer C thermoblock shaking at 300 rpm. Cells were resuspended in 2% fetal bovine serum (FBS) PBS and passed through 70-μm cell strainers (VWR, 734-2761) to obtain single-cell suspensions, which were pooled into one sample per treatment group. Cells were then incubated with DAPI (Sigma-Aldrich, D8417) and DRAQ5 (Invitrogen, 65-0880-96) for dead cell exclusion. Cells were sorted using a FACS Aria II cell sorter equipped with BD FACSDivaTM Software v6.1.3. Cells were counted and their viability was checked using the Countess 3 cell counter (ThermoFisher). Mice were randomly allocated to two treatment groups, receiving rHDL (~20 mg apoA1/kg, i.v.) or PS@rHDL (20 mg PS/kg, i.v.). Doses were administered via lateral tail vein injection. The treatment schedule consisted of 8 administrations over two weeks, during which time mice were kept on Western diet. Animals were fasted from the time of last administration until euthanasia 24 hours later, following which they were perfused with PBS and tissues of interest collected. N/A. No nucleic extraction needed as this was a single-cell transcriptomics experiment. Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).