Trypan blue dye

The trans-well (MCSP24H48, Millipore, Burlington, VT, USA) kit, composed of the upper chamber and the lower chamber, was applied to examine the permeability of the endothelial layer. First of all, 2 × 10⁵ endothelial cells (HCAECs) were seeded in the upper chamber for 24 h, with the upper chamber and the lower chamber containing a 200 μL and 600 μL culture medium, respectively. Then, the endothelial cells were treated with S100A4 recombinant proteins (4137-S4-050, R&D Systems, Minneapolis, MN, USA) or DMSO. After 24 h of treatment, the HCAEC-seeded upper chamber was washed with PBS three times. Then, the trypan blue infiltration (referring to a previous study [43 (link)]) and protein infiltration assays were applied to examine the permeability of HCAECs. In the trypan blue infiltration assay, the upper chamber was added with 100 μL of trypan blue dye (03-102-1B, Biological industries, Kibbutz Beit Haemek, Israel), and we observed how much time was needed for the trypan blue to penetrate the endothelial layer and move to the lower chamber. In a protein infiltration assay, the upper chamber was added with 100 μL or 20% FBS. After 30, 60, 90, and 120 min, we collected a 10 μL solution from the lower chamber, followed by measuring the protein concentration.

Mice were sacrificed on day 30, and serum samples were collected for antibody detection. The trachea of each mouse was cannulated, and the lung was lavaged 3 times of 1 ml normal saline. Total cell counts were determined by trypan blue dye (Biological Industries, Beit-Haemek, Israel) exclusion. The first 1 ml lavage fluid was collected and centrifuged with 1500 rpm, 5 minutes. Supernatant was collected and stored at -80°C for further analyses. Cell pellets were then resuspended into the rest of the lavage fluid. For differential cell counts, BALF cells were centrifuged (Shandon Cytospin 4; Thermo Scientific, Pittsburg, PA, USA) onto the slides and stained with Liu staining solution (Tonyar Biotech Inc., Taoyuan, Taiwan). Different cell types were distinguished under light microscopy by counting at least 200 cells based on their morphological profiles.