Csu x1 spinning disk scanner

HEK293T cells were plated on human fibronectin-coated 35 mm glass (or polymer) bottom cell culture dishes (Ibidi, Martinsried, Germany) and incubated in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% bovine serum albumin and penicillin/streptomycin. Cells were transfected with calcium phosphate (Myo10 and DCC (deleted in colorectal cancer) constructs), PolyFect transfection reagent (Qiagen) or Lipofectamine LTX (Thermo Fisher Scientific). DCC was labeled with mouse anti-DCC (monoclonal) antibodies which recognize the extracellular domain of human DCC protein (ab16793; Abcam, Cambridge, UK). The plasma membrane was stained by incubating cells with CellMask Orange (diluted 1:1000) for 5 min at 37 °C. Live-cell imaging was performed, via a Nikon Apochromat TIRF 60x/1.49 (oil-immersion) objective lens, using a spinning disk confocal microscope (UltraVIEW Vox 3D live cell imaging system coupled to a Nikon Eclipse Ti inverse microscope; Perkin Elmer, Rodgau, Germany). The system included a Yokogawa (Japan) CSU-X1 spinning disk scanner, a Hamamatsu (Japan) C9100-50 EM-CCD camera (1000 × 1000 pixels) and Volocity software.

3D images of living monocytes migrating on a 2D surface or in a collagen matrix were obtained using an UltraVIEW Vox 3D live cell imaging system (Perkin Elmer, Rodgau, Germany) coupled to a Nikon Eclipse Ti inverse microscope. The system incorporated a Yokogawa (Japan) CSU-X1 spinning disk scanner, a Hamamatsu (Japan) C9100–50 EM-CCD camera (1000 × 1000 pixels) and Volocity software. Cells were imaged via a Nikon CFI (chromatic aberration-free infinity) Apo TIRF 60x (NA 1.49) oil immersion objective lens, which has a (coverslip corrected) working distance of 0.12 mm. The temperature was maintained at 37 °C using an Okolab all-in-one stage incubator (Okolab, Ottaviano, Italy). Z-stacks of monocytes labeled with Alexa Fluor 488-conjugated anti-human CD14 antibodies were taken every 5 s or 15 s. To reduce phototoxicity during time-lapse spinning disk confocal microscopy experiments, the medium was supplemented with the reactive oxygen species scavenger N-(2-mercaptoproprionyl)-glycine (1 mM).