Torpedodna

To label endoplasmic reticulum, TGN, and mitochondria, human macrophages and CHO cells were transfected with the plasmids PR-CD5L-HA-erRFP (provided by F.X. Pimentel-Muíños, Salamanca, Spain), DsRed-galT1 (donated by M. Lorenz, Munich, Germany), and pOct-YFP (provided by K. Hell, Munich, Germany), respectively. All transfections were performed with Torpedo^DNA (Ibidi). After the transfection, cells were incubated for 30 min at 37 °C with apoptotic neutrophils or were infected for 45 min at 37 °C with Listeria in the presence of PFO/D4-GFP to visualize the association of membrane areas of increased cholesterol levels with the organelles. Subsequently, the cells were fixed, mounted in Vectashield^® with DAPI and analyzed by confocal microscopy.

Cos7 cells were seeded on 8w µ-slides (ibidi) at a density of 2–4 × 10⁴ cells per cm² and transiently transfected with plasmid DNA using Torpedo^DNA (ibidi) on the next day according to the manufacturer’s instructions. Then, 24 h post-transfection cells were stained with 1 µg ml⁻¹ Hoechst 33342 (Thermo Fisher Scientific) in PBS for 10 min to label nuclei. After staining, cells were washed three times with PBS (Sigma Aldrich) and supplemented with phenol red-free growth medium (DMEM, Sigma Aldrich) for live cell imaging. Images were acquired with a Leica DMi8 wide-field microscope (Leica microsystems) using a 100x magnification objective and the manufacturer’s LAS X 2 software. Filters: GFP (Ex.: 450–490, Em.: 500–550), DAPI (Ex.: 325–375, Em.: 435–485). Greyscale images were transformed into RGB color images and RGB merged in green or blue, respectively, with ImageJ 1.37a. RGB images were overlaid with Photoshop Version 11.0 without any further adjustments.

HEK 293T cells were cultured in 100 mm × 20 mm cell culture dishes at 4 × 10⁵ cells/dish overnight. Cells were transiently transfected with 14.7 μl of Torpedo ^DNA (Ibidi) for 24 h for a total of 5 μg of DNA/plate. On ice, after two washes with cold PBS, cells were collected by with a cell scraper and centrifuged at 80 g at 4°C during 5 min. Cell lysis and processing for co‐immunoprecipitation were done as described by either GFP‐Trap^®_A kit (Chromotek) or with the PierceTM HA Epitope Antibody Agarose conjugate (Thermo scientific).
For endogenous co‐IP, HeLa cells were cultured in 100 mm × 20 mm cell culture dishes at 1 × 10⁶ cells/dish overnight. Cells were transiently transfected and collected as described above. Cell lysis and processing for co‐immunoprecipitation were done following the manufacturers’ instructions (PierceTM HA Epitope Antibody Agarose conjugate, Thermo scientific) but using 100 μl of beads and increasing the number of washes to 5.