Facsaria 3 cell sorter system

The coding sequence of mVirD2 was enriched by PCR with an IgG secretion tag and the transmembrane domain (TM) of PDGFRB (Uniprot: P09619) and cloned in pEGFP-NI under the control of a CMV promoter. On the first day, HEK293T cells (550.000/well) were seeded in a six-well plate and cultured at 37°C 5% CO₂ atmosphere in DMEM/GlutaMAX (Gibco) supplemented with 10% FBS (Sigma Aldrich), 1% Pen/Strep (Sigma Aldrich). The next day, 2 ug/well of the purified plasmid were used for transfection with the Universal Transfection Reagent (Sigma Aldrich) according to the manufacturer's guidelines. Twenty four hours later, transfected and non-transfected cells were incubated for 2 h at 37°C with 1 μM phosphorothioate modified T1 oligo (Cy5 labeled). Cells were dissociated using either a Trypsin/EDTA solution (Thermo Fisher) or the non-enzymatic dissociation solution (Sigma-Aldrich), washed with PBS prior analysis on a FACSAria III Cell Sorter system (BD).

AML12 originally obtained from ATCC (ATCC CRL-2254) were cultured as previously described^{60 (link)}. Silencing of Scarb1 was performed by transfecting 100 nM smart pool to Scarb1 or a mock control (Horizon) with RNAimax reagent (Invitrogen) in OptiMEM (Invitrogen). Expression of SR-BI and SR-BII was obtained by cloning codon-optimized SR-BI and SR-BII (generated by gblock synthesis at IDT) into pLV (PGK)-GFP Neo vector. Third generation lentiviruses were generated in human embryonic kidney-293T cells (ATCC CRL-1573) and purified by high-speed centrifugation. Viruses were resuspended in media supplemented with polybrene and added to the cells. When indicated, cells were incubated with 100 ng ml⁻¹ Dil-HDL for 4 h and lipid uptake was quantified by FACS using a FACSAria III cell sorter system (BD Biosciences).