Cell observer live imaging microscope

Cells were plated in 24-well tissue culture dishes with ibidi® culture inserts (ibidi LLC, Verona, WI, USA) at 80–90% confluence. The following day, the culture inserts were detached to form a cell-free gap in the cell monolayer and the medium was changed to remove floating cellular debris. Migration was monitored using a Zeiss Cell Observer Live Imaging microscope (Zeiss, Thornwood, NY) coupled with a CO₂ and temperature-maintenance system. Time-lapse images were acquired every 30 min during 24 h using a Zeiss AxioCam MRc camera. The area of each individual wound gap was measured in each culture condition using Image J image analysis program. Cell migration was analyzed by calculating the difference between the initial scratch area and the residual gap after 5, 15 or 20 h for UT-SCC-42B, FaDu or CAFs, respectively. For each culture condition, cell migration was quantified by comparing the area of the cell-free gap after normalization to the control cells.

Cells were suspended in DMEM plus 5% Methyl cellulose (Sigma) at 80,000 cells/ml medium to form cell spheroids (2,000 cells/spheroid) by serial pipetting of 25 μl into a non-adhesive Petri dish, and incubated in an inverted position for 18 h. Next day, each cell spheroid was transferred to an individual well of 96-well plate and embedded into bovine collagen matrix (Advanced Biomatrix PureCol), and filled with 100 μl of complete media. Collective cell invasion was monitored using a Zeiss Cell Observer Live Imaging microscope (Zeiss, Thornwood, NY) coupled with a CO₂ and temperature-maintenance system. Time-lapse images were acquired every 30 min during 20 h using a Zeiss AxioCam MRc camera. The invasive area was determined by calculating the difference between the final area (t = 20 h) and the initial area (t = 0) using image J analysis program, and data were normalized to the control cells.

MDA-MB-231 and MCF-7 cells were suspended in DMEM medium plus 5% methyl cellulose (Sigma-Aldrich, Madrid, Spain) at 80,000 cells/mL. Cell spheroids were subsequently formed by serial pippeting of 25 μL into a non-adhesive petri dish (2000 cells/spheroid) and incubated in an inverted position for 18 h. Next, each spheroid was transferred to an individual well of 96-well plate and embedded into a volume of 110 μL of 2.3 mg/mL bovine collagen type I matrix (PureCol, San Diego, CA, USA) from advanced Biomatrix, and filled with 100 μL of complete media.
Collective cell invasion was monitored using a Zeiss Cell Observer Live Imaging microscope coupled with a CO₂ and temperature maintenance system. Time-lapse images were acquired every 20 min during 24 h using a Zeiss AxioCam MRc camera (Carl Zeiss, Sliedrecht, The Netherlands). The area of each individual spheroid was measured using ImageJ Fiji analysis program (version 1.50i, Bethesda, MD, USA). The invasive area was determined by calculating the difference between the final area (at each represented time) and the initial area (t = 0 h), and data were normalized to the control (vehicle-treated) cells. Each assay was performed at least three times using quadruplicates.