Immersol w

Live cell-imaging was conducted with a Leica SP5 microscope (Leica-Microsystems, Germany). Cells were maintained at 37°C in an atmosphere containing 6% CO₂/94% air using a thermostatically controlled heated enclosure with passive humidification and a separate gas mixing unit (Life Imaging Services, Switzerland). Images were collected using a 40×, 1.1 numerical aperture, water immersion objective (Leica-Microsystems, Germany). Immersol W (Zeiss, UK) with a refractive index of 1.334 was used as a water-substitute immersion fluid for long-term imaging. Simultaneous RCM and brightfield images were acquired with separate photomultiplier tubes (PMT) using the Argon 488 nm laser line. The RCM signal was collected between wavelengths of 478–498 nm with a pinhole size of 57 μm. Images were collected with a line scanning speed of 600 Hz at 1024×1024 pixel resolution. A line and frame average of 2 was applied. A total of 34 fields of view per condition were followed by time-lapse microscopy. Representative fields of view were selected from structures formed by single cells. For the quantitative analysis of fiber organization, structures from 0% (n = 12), 5% (n = 11), and 50% (n = 10) Matrigel were analyzed at each hour between the first 3–12 hours following cell seeding. At day 5, morphology and fiber organization were quantified for 19 independent structures in 5% Matrigel.

Live cell microscopy was performed on the inverted confocal laser scanning instrument Zeiss LSM780 (Zeiss, Oberkochen, Germany) to visualize binding and internalization of FAM-labeled peptides. Cells were seeded at a density of 1.5 × 10⁴ per well in an 8-well µ-slide (Nunc, LabTek, Thermo Fisher, Karlsruhe, Germany) in 300 µL RPMI medium and incubated overnight. Microscopy was performed while maintaining cell culture conditions in an incubation chamber at 37 °C. Cells were incubated with 10 µL NucBlue Live ReadyProbes Reagent (Invitrogen) and 50 nm Lysotracker DND-99 (Invitrogen) for 10 min. Peptides were diluted to 100 µM stock solutions in DMSO and added to the cells with 5 µM concentration for 10 min at 37 °C (final DMSO concentration 5%). Residual peptide was removed with five extensive washing steps with RPMI (w/o phenolred and FCS) before imaging the cells with a 63× objective (LCI Plan_neofluar 63×/1.3 Imm Korr DIC M27) using immersion oil (Immersol W (2010), Zeiss). Laser and detector ranges were used with corresponding main beam splitters as given in Table 1. Image acquisition was performed using Zeiss Zen 2011. Image analysis was carried out using ImageJ (National Institute of Mental Health, Bethesda, MD, USA). A macro was used to analyze areas of colocalization between the carboxyfluorescein signal of the FAM-labeled peptide and the Lysotracker DND-99 signal.