The RPMI 2650/CCL-30 (ATCC) nasal epithelium cell line was cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum. Cells were incubated in a humidified incubator at 36 °C in an atmosphere containing 5% CO2. Cells were only used for experiments at passages 5–10. Cells were transfected by administering silencing RNA (siRNA) for MMP3, MMP7, CTSK, or control siRNA (Qiagen, Gaithersburg, MD). Cells were stimulated with 1 μg/ml S100A9 recombinant protein (Novus Biologicals).
Cell proliferation was measured by counting the cells in the logarithmic phase using Cell Counting Kit-8 (#CK04; Dojindo Kumamoto, Japan) as previously outlined22 (link). The cells were first transfected with siRNA or plasmid and then were plated into a 96-well plate. Cells from each group were plated in 3 wells, and each well contained 2 × 103 cells. The absorbance of each well was measured with a reader at the same time over 3 consecutive days. This process was repeated in triplicate for the statistical analyses. For confirmation, the BrdU proliferation assay (BD Biosciences, San Jose, CA, USA) was performed according to the manufacturer’s instructions. The stained cells were analyzed in on the Guava easyCyte flow cytometer from Millipore.
Cell proliferation was measured by counting the cells in the logarithmic phase using Cell Counting Kit-8 (#CK04; Dojindo Kumamoto, Japan) as previously outlined22 (link). The cells were first transfected with siRNA or plasmid and then were plated into a 96-well plate. Cells from each group were plated in 3 wells, and each well contained 2 × 103 cells. The absorbance of each well was measured with a reader at the same time over 3 consecutive days. This process was repeated in triplicate for the statistical analyses. For confirmation, the BrdU proliferation assay (BD Biosciences, San Jose, CA, USA) was performed according to the manufacturer’s instructions. The stained cells were analyzed in on the Guava easyCyte flow cytometer from Millipore.