Transwell inserts with 8 μm pores

Harvested BAL macrophages were counted, and a migration assay was performed using transwell inserts with 8-μm pores (VWR). Briefly, 3 × 10⁵ macrophages were added to the top chamber, and 5 × 10⁶ CFU of S. pneumoniae in RPMI 1640 supplemented with 5% fresh mouse serum was added to the bottom chamber. Cells were allowed to migrate for 6 h (37°C, 5% CO₂). Transwell inserts were fixed, permeabilized with methanol, and stained with methylene blue. Cells on the input side of the membrane were scraped off, and membranes were mounted on microscopy slides. Results are presented as the average of cell counts in 5 random high power (100×) fields of view.

Transwell migration [67 (link)] and invasion assays [16 (link)] were performed as described previously. The Transwell inserts with 8 μm pores (VWR, West Chester, PA) were either left uncoated for migration assay or coated with 40 μl Matrigel (Corning Life Sciences, Corning, NY). Serum-deprived cells in 0.1% serum-containing media were plated in the top chamber for 2 h to allow attachment. 100,000 cells were used for migration assays while 50,000 cells were used for invasion assays. Either 0.1% or 10% serum-containing medium was then added to the bottom chamber. After 48 h, cells were washed, fixed in methanol at – 20°C, and stained with propidium iodide. Cotton swabs were used to remove cells from the top chamber. Fluorescence images of migrated or invaded cells were processed using the ImageJ software to quantify the number of cells. All conditions were performed in triplicates and four high-power fields were imaged per replicate.