Dotap transfection reagent

Human PBMCs were isolated by Ficoll-gradient centrifugation, washed, and counted. PBMCs were incubated with anti-human CD14 MicroBeads (Miltenyi Biotec), and monocytes were separated on a LS magnetic column on a QuadroMACS separator (Miltenyi Biotec). Cells were differentiated with human GM-CSF (50 ng/ml, Sino Biological) in RPMI 1640 medium with L-glutamine (300 mg/L, ThermoFisher), supplemented with 10% heat-inactivated fetal bovine serum (FBS, ThermoFisher) and 1% penicillin/streptomycin (Gibco) for 7 days followed by 24 h of native HDL (1 mg/ml) treatments in FBS-free media. Bone marrow cells were isolated from WT C57BL/6 mouse femurs and differentiated to BMDMs using murine GM-CSF (50 ng/ml, Tonbo Biosciences) in Dulbecco's modified Eagle's medium (ThermoFisher) supplemented with sodium pyruvate (110 mg/L), L-glutamine (584 mg/L), 10% heat-inactivated FBS (ThermoFisher), and 1% penicillin/streptomycin (Gibco) for 7 days. BMDMs were transiently transfected with tDR-GlyGCC-30-AF647 (1 μg/ml) complexed to DOTAP transfection reagent (Roche) for 24 h in serum-free media followed by native HDL (1 mg/ml) treatments in FBS-free media for 24 h.

A transfection/infection assay [101] (link) was performed to examine the ability of the recombinant bacmids to infect Sf9 cells. Bacmid DNA was extracted from E. coli cells in strict accordance with the Bac-to-Bac manufacturer's instructions (Invitrogen Life Technology). The transfection of bacmid DNA into insect cells was performed using the DOTAP transfection reagent (Roche Applied Science). Briefly, 5 µl of bacmid DNA solution and 8 µl of DOTAP were mixed with 35 µl and 72 µl of HBS buffer, respectively, and gently shaken. The two solutions were then combined, mixed by pipetting, and incubated for 30 min at room temperature. The DNA-DOTAP complex was diluted with 360 µl of TC100 serum-free medium and layered onto a monolayer of Sf9 cells at approximately 50-60% confluence in 35-mm Petri dishes. After 5 h of incubation, the DNA-DOTAP-containing supernatant was replaced with fresh medium, and the incubation was continued. The transfected monolayers were inspected with a fluorescence microscope at 48 and 96 h p.t. to determine the percentage of cells emitting fluorescence. At 96 h p.t., the cells were harvested for DNA extraction, and the supernatants were harvested and used for the inoculation of fresh monolayers. The inoculated monolayers were examined with a fluorescence microscope at 96 h p.i., and the cells were harvested for DNA extraction.