Diff quick staining kit

A549 cells were incubated in a 24-well plate for 24 h. After serum starvation for 24 h, cells were treated with TGF-β1 and sunitinib/cRGDfK or combination for 48 h. Collected cells in culture medium containing 5% FBS was added to the bottom of a Boyden chamber. After placing over the gelatin-coated membrane filter, the silicone gasket, and the top chamber, the cell suspension was added to the top chamber, followed by incubation at 37°C in 5% CO₂ for 6 h. The membrane filter was stained using the Diff-Quick staining kit (Dade Behring) and filter was dried and stabilized on a glass slide using 30% glycerol solution, the migrated cells were counted in three randomly selected fields at 400× magnification. This experimental procedure is referred to our previous study [18 (link)].

The Matrigel invasion assay was performed according to the manufacturer's instructions (BD Biosciences). Briefly, 1×10⁴ cells in 0.5 mL of medium containing 1% FBS were added to the transwell insert, which was seated in 750 μL of complete medium (10% FBS). After a 24-h incubation, noninvading cells were mechanically removed. Cells that had migrated through the Matrigel were stained with the Diff-Quick staining kit (Dade Behring). Cells were counted in five representative microscopic fields (×200 magnification) and photographed.

Bronchoalveolar lavage was performed after removal of the lungs post mortem. The trachea was cannulated with a 20-gauge blunted stainless steel catheter. Serial 1 ml lavage samples were taken x10 with ice-cold HBSS (no Ca²⁺ or Mg²⁺) containing 1 M HEPES, penicillin/streptomycin, and 0.5 M EDTA. The lavage fluid was centrifuged at 300×g for 5 minutes at 4°C, and cell pellets (>99% alveolar macrophages) were preserved. If used for primary culture, cell pellets were resuspended in serum-free RPMI1640 medium. The primary murine macrophages were seeded into 24 well plates and incubated overnight at 37°C in a humidified atmosphere containing 5% CO₂. The cells were stimulated with 0.5–1 ug/ml LPS (Serotype 0111:B4, Sigma) or E. coli, 10⁶ CFU for pre-specified time points. Supernatants were collected for protein analysis and cells were collected in Trizol (Life Technologies) for RNA analysis. For BAL cytokine measurement, the first 1 milliliter lavage sample was saved separately for protein analysis. BAL cell counts were performed as previously described [52] (link), [55] , with cytocentrifuged slides stained with the Diff-Quick staining kit (Dade Behring) after counting suspended cells using a hemocytometer.