Isoton

After BALF collection, lungs were perfused with ISOTON (Beckman Coulter) to flush the vascular compartment. The lung inferior and post-caval lobes were processed using a Precellys tissue homogenizer (Bertin Instruments) in 1 ml phosphate-buffered saline (PBS) with complete Protease Inhibitor Cocktail (Roche). The extracts were centrifuged for 10 min at 9,000 g and supernatants stored at -80°C for ELISA or western blot assays.

After BAL the lung were perfused with Isoton^® (Beckman Coulter France, Villepinte) to flush the vascular content. For protein analysis, the trilobed lung part was homogenized by a rotor-stator (Ultra-turrax^®) in 1ml of T-Per protein extraction buffer (ThermoFisher Scientific™) mixed with protease and phosphatase inhibitor (ThermoFisher Scientific™). The extract was centrifuged 10 min at 10000 rpm and the supernatant was stored at -80°C before mediator measurement and immunoblotting analysis.

The sorting process occurred in a microfluidic chip, which included a sorting area and a filtration unit. The sorting area contained a sample inlet channel with a section area of 150 × 50 μm. There were four other channels with 50 × 200 μm for injecting buffer and passing cells. The filtration unit was constructed with a 5 × 5 μm slit-filter to capture cells (for example, MCF-7 with a diameter ∼25 μm). The microfluidic chip was fabricated by one-step replica moudling into polydimethylsiloxane under a patterned silicon master and then sealed to a glass substrate via plasma oxidation. The Pdot-labelled MCF-7 cells in 1 × PBS with 10 wt % BSA were injected into the microfluidic chip by a syringe pump. Isoton from Beckman Coulter Inc. (Chino, CA, USA) was used as the buffer. The fluorescence signal of cells excited by a 488-nm laser beam was collected by fiber-coupled avalanche photodiodes from Excelitas Technologies (Waltham, MA, USA) with a filter of 570/20 nm. The sorting process of eDAR was automatically controlled by an in-house LabVIEW (National Instruments, Austin, TX, USA) script and a field-programmable gate array device built in-house. The sorting threshold was set based on the signal discrepancy between ON-state and OFF-state cells. The hydrodynamic sorting was controlled by a solenoid (INKA1226212H) purchased from the Lee Co. (Westbrook, CT, USA).

Lungs were perfused with Isoton (Beckman Coulter) to flush the vascular content. Washed lungs and ileum were homogenized by a rotor-stator (Ultra-turrax) in PBS with protease inhibitor cocktail (Roche) for mediator measurement or in RIPA buffer with protease inhibitor cocktail (Thermo Fisher Scientific) for immunoblotting analysis. Extracts were centrifuged and supernatants stored at –80°C.

After BAL the lungs were perfused with Isoton® (Beckman Coulter France, Villepinte) to flush the vascular content. Lungs were homogenized by a rotor-stator (Ultra-turrax®) in 1 ml of PBS for ELISA dosage. The extract was centrifuged 10 min 10,000 rpm and the supernatant was stored at −80°C before mediator measurement.