Cim plate 16

The xCELLigence® DP system (Roche Diagnostics GmbH, Germany) was used to study migration as previously described [25 (link)]. Briefly, AGS-Gr cells were seeded into (5.0 x 10⁴ cells/well) the CIM-Plate 16 (Roche). The lower chamber contained 1 nM gastrin alone or in combination with ULK1 inhibitor SBI-0206965 (10 μM) final concentration) (Apex Biosciences A8715), Compound C (Millipore), HCQ (20 μM), BafA1 (100nM). Cell migration was monitored every 15 min on a RTCA DP instrument for 24 h. Data analysis was carried out using RTCA Software 1.2 supplied with the instrument.

Cell migration was determined using the xCELLigence System (Roche) with the CIM-Plate 16 and RTCA DP Instrument (Roche) according to the manufacturer's instructions. Briefly, 40,000 cells were added to the upper CIM-Plate 16 chamber in media containing 0.2% fetal bovine serum (FBS). Media with 10% FBS was added to the lower CIM-Plate 16 chamber and the CIM-Plate 16 was incubated in the RTCA DP Instrument for 48 hrs. Cell migration as a function of real-time changes in electrical impedance was monitored by the xCELLigence System. Cell Index (referred to here as Migration Index) and standard deviation of replicates were calculated using the supplied RTCA Software (Roche).

Cell migration experiments were conducted in the xCELLigence RTCA DP system (Roche). The cells were suspended in serum-free medium and seeded in the upper chambers of 16-well CIM-Plate 16 plates (40,000 cells in 150 μl medium/well; Roche). Regular medium with 10% FBS was added to the lower chamber of the CIM-Plate 16. The experiment setting and plate design were similar to those of conventional Transwell migration assays. The cell index, which is proportional to the number of cells that migrate through the pores of the upper chamber, was recorded in real-time every 30 min for up to 24 h. The average cell migration index was calculated from at least four replicate experiments, and is presented as mean ± SE.

The xCELLigence DP Real-Time Cell Analyzer (RTCA) equipped with a CIM-plate 16 (Roche) was used to monitor cell proliferation, migration and invasion. For the invasion experiments, the membrane was coated with Matrigel (BD Biosciences). For the migration assays, the membrane was left uncoated. Cells (5×10³) were starved in a serum-free media for 8 h. 10% FBS in lower chamber was used as a chemoattractant. Migration and invasion was monitored every 30 min for 48 hours with different concentrations of TTB treatment.
Invasion and migration assays were also performed as described previously [28 (link)]. Briefly, on 24-well plates, A549 or 4T1 cells (2×10⁴) were placed in 0.2 ml serum-free media onto the upper chamber of regular or Matrigel-coated transwell filters. The lower chamber was filled with 0.6 ml DMEM supplemented with 10% FBS. After 18-36 h of incubation with sTGF-βRIII or pan TGF-β mAb (1D11) or TTB in the presence or absence of 20 pM TGF-β1, the cells on the upper surface of the filter were removed, and cells underneath of the filter were fixed and stained with a 0.02% crystal violet solution. The number of cells were counted in three fields using a Nikon ECLIPSE Ti microscope at 100 × magnification.

The chemotaxis assay was carried out as described elsewhere^{66 (link)}. Briefly, cell invasion and migration plates (CIM-Plate 16; Roche) were coated with 20 μg/ml fibronectin (Sigma) in PBS at 37 °C, 30 min, then washed twice with 50 μl PBS. The lower chambers were filled with 160 µl of supernatants from MAC-T cells infected with the relevant strain of S. aureus for 24 h. Upper chambers received 35 µl DMEM with 1% FBS. After 1 h at room temperature, background impedence was obtained at 37 °C, 5% CO₂. To the top chamber of each well, 100 µl of neutrophils (1.2 × 10⁷ cells/ml in DMEM with 1% FBS) was added and impedence measured every 5 min for 7 h. Several controls were included: (i) positive control: neutrophils in upper chamber, 150 ng/ml bovine IL-8 (Kingfisher Biotech Inc) in lower chamber (ii) negative control: neutrophils in upper chamber, 0 ng/ml IL-8 in lower chamber (iii) chemokinesis: neutrophils in upper chamber, 150 ng/ml IL-8 in upper and lower chambers (iv) fugetaxis: neutrophils and 150 ng/ml IL-8 in upper chamber, medium only in lower chamber; (v) uninfected cells: neutrophils in upper chamber, medium from cells uninfected with S. aureus in lower chamber. The slope of the exponential part of the curve (1 h) was calculated using RTCA software 1.2.1.