Ibitreat eight well chambers

Human fibroblasts were cultured in ibiTreat eight‐well chambers (ibidi) for live‐cell imaging overnight prior to treatments. Cells were treated with 30 μM agonist in DMSO (to a final DMSO concentration of 0,3%) overnight or up to 48 h, and the lactosylceramide trafficking assay was subsequently initiated: Cells were washed once with PBS, and 25 μM LacCer (BODIPY FL C5‐Lactosylceramide, Invitrogen) pulsed in serum‐free culture medium for 1 h at 37°C. Cells were washed twice with PBS and chased with complete DMEM (including 15% FBS and the indicated agonists) for 2 h at 37°C. LyTr‐DR (LysoTracker‐Deep Red; diluted 1:10,000, Invitrogen) was added 1,5 h into the chase time to visualize acidic organelles. The cells were subsequently washed three times with PBS, before adding a complete phenol‐red‐free medium for imaging. The cells were transferred to a pre‐heated 37°C incubation chamber mounted onto a Zeiss Confocal microscope (LSM 880) and imaged using a 63 X water objective at 488 nm (LacCer) and 633 nm (LyTr) excitation wavelength, respectively. For data quantification, the Fiji software was used alongside the JACoP plugin for colocalization quantification, calculating the Mander's coefficient for LyTr‐DR overlapping LacCer. LacCer density calculations were performed using Harmony High‐Content Imaging and Analysis Software (PerkinElmer).

NIH/3T3 cells were seeded at a density of 2 × 10⁴ cells/well of a 96-well plate and allowed to attach overnight. On the next day, cells were treated with increasing concentrations of TAT-I24 and infected with MCMV strain delm157-luc rep with centrifugal enhancement twice at 800× g for 15 min. After 48 h, cells were fixed with 4% formaldehyde and stained with 1% crystal violet solution for 30 min at room temperature followed by three times washing with water. Microscopic examination was performed using a Live Cell Video Microscope (Leica Microsystems; Wetzlar, Germany).
NIH/3T3 cells were seeded at a density of 4 × 10⁴ cells/well into ibiTreat eight-well chambers (ibidi, Gräfelfing, Germany). Bromodeoxyuridine (BrdU)-labelled virus was adsorbed to cells in the absence or presence of 10 µM TAT-I24 as described before [16 (link)]. Pepsin treatment was performed by incubation of cells after fixation, permeabilization and denaturation with Pepsin Reagent, Ready to Use, Antigen Retriever (Merck; Schnelldorf, Germany) for 20 min. Cells were then washed twice with 1xTBE followed by three washes with PBS. Staining with BrdU-antibody was performed as described earlier [16 (link)].