Dm irb microscope system

MDA-MB 231 cells were plated into 24-well plates (10⁵ cells/well) in complete medium. Confluent monolayers were nutrient starved by growing them for 24 h in medium containing 0.5% FBS and then scratched using a 200 μl pipette tip. After washing, cells were treated or not with 10 ng/ml of refp17 or vp17s in complete medium (10% FBS). When reported, starved MDA-MB 231 cells were pretreated with 2.5 μg/ml of mAb to CXCR1 (mAb 330; R&D, Minneapolis, MN, USA) or to CXCR2 (mAb 331; R&D), or with an isotype-matched mAb (2.5 μg/ml; R&D) for 1 h at 37 °C before proteins stimulation. In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways. Cell migration was evaluated at different time points using an inverted microscope (DM-IRB microscope system, Leica, Buffalo Grove, IL, USA). Cells were photographed using a CCD camera (Hitachi Inc., Krefeld, Germany). Wound closure was monitored over 12 h. In some experiments, in order to count the cells migrating into the wound area or protruding from the border of the wound, cells were fixed before wound closure and stained with Comassie brilliant blue.

Microchannels were fabricated using standard techniques of soft photolithography in polydimethylsiloxane (PDMS). All channels were 100 μm(w) × 22 µm(h) containing a single 100(lc) × 20(wc) µm constriction. Cells were centrifuged at 300 g for 1 minute at room temperature, resuspended in PB2, and diluted in physiological salt solution (PSS, 4.7 mM KCl, 2.0 mMCaCl₂, 1.2 mM MgSO₄, 140.5 mM NaCl, 21 mM Tris, 11.1 mM dextrose, pH7.4) containing 0.1% BSA. High-speed videos were acquired at ~1900 frames/sec with a 30μs exposure with a Phantom Miro M120 camera (Vision Research) on a DMIRB microscope system (Leica) with a 63 × / oil immersion objective. Movies were analyzed using ImageJ⁴¹ . At least three independent experiments were performed and more than 30 cells were analyzed at each gestational age.

A total of 25,000 PCs were detached from the flasks and resuspended in a mixture of collagen I, 1X DMEM/F-12 medium, and 1X sodium bicarbonate solution. Gel drops were then created in a 4-well plate (Nunc., Thermofisher Scientific) and incubated at 37 °C for 1 h. After incubation, cell-loaded collagen drops were bathed in a medium containing FGF-2 (50 ng/mL) (Santa Cruz Biotechnology) or VEGF-A (50 ng/mL) (Miltenyi Biotec). Collagen drops bathed in EBM-2 media with 0.5% serum or EGM-2 media with 10% FBS were used as a negative and positive control, respectively. PCs migration outside collagen drops was quantified as the distance covered at least after 72 h obtained with a DM-IRB microscope system (Leica, Wetzlar, Germany) and photographed with a Hitachi KP-D50 camera. The distance was measured manually as a parameter of length from the edge of the collagen drops to the leading edge of the cells.