Giemsa

The cell migration used a transwell chamber with a polycarbonic membrane (6.5 mm diameter and 8 μm pore size; Corning). The serum-free DMEM of no-transfected or transfected cell was added to the upper chamber, and 600 μl of 15% FBS–DMEM was added to the lower chamber. The cells that were migrated to the lower chamber surface were fixed with 4% methanol and stained with 10% Giemsa (Solarbio, China), and the unmigrated cells on the membrane were wiped off with cotton swabs.

Airway inflammation was assessed by differential count of inflammatory cells in bronchoalveolar lavage (BAL) fluid. For collection of BALF at 2, 6, and 24 hours after ACS exposure, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). After partial excision of the trachea, a plastic cannula was inserted, and the airway was washed twice with 0.5 mL phosphate-buffered saline (PBS) at 37°C. The rib cage was then gently massaged to maximize the number of cells carefully collected by aspiration. This procedure was performed 6 times. The saline lavage was centrifuged at 200 × g at 4°C for 10 min. The supernatants were decanted, and the cell pellets were resuspended in 300 μl of saline, kept at 4°C until cell counting using PlasMET counting chamber slides. The differential inflammatory cell counts in BALF were calculated using standard morphological criteria in the representative slide (minimum of 300 cells per slide) which were stained with Giemsa (Solarbio, Beijing, China) according to manufacturer's instructions.

EJ and UMUC3 cells (600 cells per well) were added to 6-well plates and cultured for 14 days. The resultant colonies were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with Giemsa (Solarbio, Beijing, China). The colonies were photographed with an inverted microscope (Axioskop 2 Plus; Zeiss, Germany) and counted.