Gelatin coated 8 well μ slides

HUVECs were seeded in gelatin-coated 8 well μ-slides (ibidi GmbH, Martinsried, Germany) at a density of 5×10³ cells per well. Cells were serum-starved for 24 hours before stimulation with PZ (3 µg/ml) or SDF-1 (50 ng/ml) in serum- and growth-factor reduced medium (containing 5% FCS). After 8 and 24 hours of stimulation cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and blocked with 1% bovine serum albumin in PBS for 1 hour. Immunofluorescence staining was performed with primary antibody anti-CXCR4 (1∶50, MAB21651, R&D Systems) over night at 4°C followed by incubation with secondary antibody goat-anti-rat-Alexa⁵⁵⁵ (1∶400, Life Technologies GmbH, Darmstadt, Germany) for 1 hour at room temperature. Samples without primary antibodies served as negative control. Additionally, nuclei were stained with DAPI (1∶1000; AppliChem, Darmstadt, Germany) for 10 min at room temperature. The fluorescence signals were visualized by using a confocal laser scanning microscope (LSM 780 ELYRA PS.1 microscope, Carl Zeiss Microscopy GmbH, Jena, Germany).

HFFs were seeded on two gelatin-coated 8-well μ-slides (Ibidi) at a density of 4 x 10⁴ cells per well. Cells were precooled on ice for 15 min and medium was subsequently exchanged against ice-cold, freshly produced cell-free infectious supernatant of TB40/E. Attachment of viral particles was allowed on ice for 1 h. Supernatant was exchanged against precooled MEM5 with or without 200 ng/ml PDGFR-alpha-Fc and incubated on ice for 2 h. One of the plates was shifted to 37°C for 2 h, whereas cells of the replica-plate were treated with prewarmed 50% polyethylene glycol 1500 PEG; Roche) for 30 sec. The PEG was immediately removed by five-times washing with prewarmed PBS and cells were incubated in prewarmed MEM5 with or without 200 ng/ml PDGFR-alpha-Fc for 2 h at 37°C. Medium was exchanged to MEM5 on both replica plates and infection was allowed to proceed for 24 h before infection efficiencies were assessed by staining of viral IE antigens.