Aim software

3D image z-stacks were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss, Thornwood, NY, USA) using either a Plan-Neofluar 10×/0.3, Plan-Neofluar 40×/0.75 or C-Apochromat 40×/1.2 W objective (Carl Zeiss, Thornwood, NY, USA). The EGFP reporter was excited with the 488 nm laser line using the FITC filter and all other imaging parameters were as described in Kasemeier-Kulesa et al. (2005) (link). Images were collected, processed and analyzed using AIM software (Carl Zeiss, Thornwood, NY, USA). Statistical analysis was performed using the Student's t-test.

Pulmonary airway cells in MCF‐7 medium were diluted to 1000, 500, 250, 100, or 50 SAEC per well of a 96‐well tissue culture plate in duplicate wells. SAEC were given 24 h at 37^°C to attach. Following incubation, MCF‐7 medium in each well was removed and replaced with 2 × 10³ MCF‐7 cells in 0.1 mL of medium. After 24 h at 37^°C, cells were stained with 2% crystal violet and scattering was measured as described above.
For visualization of MCF‐7 interactions with pulmonary epithelium, SAEC were labeled with 1 μg/μL SNARF^®‐1 carboxylic acid, acetate succinimidyl ester (Invitrogen, Thermo‐Fisher Scientific, Waltham, MA) for one hour, followed by transfer of the cells in SAGM onto glass chamber slides at approximately 50% confluency. After overnight incubation to allow the cells to adhere to the glass, GFP‐labeled MCF‐7 cells were added in 50% SAGM and MCF‐7 medium. The use of the vital dye and GFP allowed imaging of living cells. After another overnight incubation, cells were visualized and photographed with a Zeiss LSM510 fluorescent microscope, with AIM software (Carl Zeiss MicroImaging, Inc., Jena, Germany). Excitation and transmission settings for GFP were 488 and 505 nm, respectively, and settings for SNARF^®‐1 carboxylic acid, acetate succinimidyl ester were 488 and 560 nm, respectively.