Perm wash buffer

Briefly, after polarization in T75, M1 and M2 were collected and stained for surface markers during 10 min at 4 °C in DPBS/FBS 5% using CD86–APC (anti-human, monoclonal recombinant IgG1, REA968, Miltenyi Biotec, Leiden, The Netherlands); CD163–APC (anti-human, monoclonal recombinant IgG1, REA812, Miltenyi Biotec, Leiden, The Netherlands); and CD206–APC (anti-human, monoclonal recombinant IgG1, DCN228, Miltenyi Biotec, Leiden, The Netherlands) at a dilution of 1/50. For intracellular staining, cells were first fixed and permeabilized for 20 min at 4 °C with Cytofix/Cytoperm (BD Biosciences, Erembodegem, Belgium) and then washed with PermWash buffer (BD Biosciences, Erembodegem, Belgium). Next, cells were stained for 10 min at room temperature with CD68–PE antibody (anti-human, monoclonal recombinant IgG1, REA886, Miltenyi Biotec, Leiden, The Netherlands) diluted 50× in PermWash buffer. After washing, the cells were resuspended in DPBS/FBS 5% and analyzed by flow cytometry. The samples were assessed in BD LSRFortessa X-20 and the data was analyzed using FlowJo_v10.7.1.

Lentiviral vectors expressing shRNA targeting apoE or control shRNA were produced in HEK-293T cells. Huh-7.5 cells were transduced with lentiviral vector. 24 h post-transduction, cells were transfected with plasmid allowing tecVLPs production or infection of HAZV. 72 h post-transfection, supernatants were harvested and used for assessment of transduction efficiency or infectivity and RNA levels. Level of KD was checked by apoE intracellular FACS staining. Cells were fixed and permeabilized with Cytofix/CytoPerm (BD Biosciences) according to manufacturer instructions. Cells were incubated with primary antibody (AHP2177, 1/2000) diluted in Perm/Wash buffer (BD Biosciences) for 1 h at 4 °C with regular checking. After three washes with Perm/Wash buffer, cells were incubated for 1 h at 4 °C with secondary antibody. Cells were washed three times with Perm/Wash buffer before resuspension in PBS and flow cytometry acquisition (MACSQuant® VYB Flow Cytometer; Miltenyi Biotec). Data were analyzed with FlowJo software (BD Biosciences). The gating strategy is depicted in the Supplementary Fig. 4.