Albumax 1 lipid rich bsa

After electroporation of PLC3 cells with p6-wt, p6-V1 or p6-V2 RNA or PBS, 1 × 10⁶ cells were seeded into T-75 flasks in DMEM complete medium. Eight hours after seeding, the medium was changed to HEV medium: DMEM/M199 (1v:1v), 1 mg/ml of lipid-rich albumin (AlbuMAX™ I Lipid-rich BSA, ThermoFisher Scientific), 1% non-essential aa and 1% pyruvate sodium (ThermoFisher Scientific). Then, the HEV producing cells were incubated at 32°C and 5% CO₂ for 10 days.
Next, Huh-7.5 cells seeded into coated 96-well plates (8,000 cells/well) were infected with undiluted and serially diluted supernatants from HEV-producing cells. The inoculum was removed after 8 h and cells were overlaid with fresh medium. Three days post-infection at 37°C and 5% CO₂, cells were fixed and processed for indirect immunofluorescence with anti-ORF2 antibody 1E6 (Millipore). For confocal microscopy analysis, Huh-7.5 cells were seeded on glass coverslips in 24-well plates and infected with 500 μl of undiluted infectious cell supernatant.

AlbuMAX I Lipid-Rich BSA and RPMI-1640 were purchased from Thermo Fisher Scientific (Leicestershire, UK). Dolichol and dolichyl-P 13–21 were purchased from Avanti (Alabama, USA). [1-(n)-³H] GGOH (14 Ci/mmol; 1 mCi/mL), [1-(n)-³H] FOH (14 Ci/mmol; 1mCi/mL) and L-[4,5-¹⁴C (U)] isoleucine (200–300 mCi/mmol; 0.1 mCi/mL) were purchased from American Radiolabeled Chemicals (St. Louis, USA). SYBR Green I nucleic acid gel stain and SYTO 11 were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, EUA). Sterile stock solutions were prepared at 10 mM for fosmidomycin sodium salt hydrate in water, 2 mM clindamycin hydrochloride in water, 125 mM of GGOH in ethanol and 200 mM of each other non-radiolabelled prenols in ethanol. All other reagents were purchased from Sigma (St. Louis, Missouri USA) or specific companies, as cited in the text. Polyprenyl phosphates were obtained by mild acid treatment of the respective commercial pure pyrophosphates (Sigma; Sigma codes F6892 and G6025) [53 (link)]. For this work, a Cre-LoxP P. falciparum NF54 strain [54 (link)], a generous gift of Moritz Treeck (The Francis Crick Institute, London, United Kingdom), was employed. Erythrocytes and CPDA-1 treated hospital-grade plasma bags for transfusions (AB+) were obtained from the Blood Center of Sírio Libanês Hospital (São Paulo, Brazil).