Fbs dmem

AEC2 cells were isolated from murine or human lungs as previously described with slight modifications.⁵⁹ (link) Briefly, Sftpc-Cre-ERT2-human-IGFBP2flox transgenic mice and corresponding littermate’s mice were sacrificed after 14 days of bleomycin treatment, and the lung was perfused with 30 ml of saline to remove the blood. Either murine or human lungs were digested using mixture of 1 mg/ml of collagenase I and 5 U/ml of Dispase at 37°C for 25 minutes. Lung cell suspensions were passed through 400 μm filters (Pluriselect, USA) and neutralized by equal volume of 20% FBS/DMEM (Invitrogen, USA) containing 200 U/mL DNase (Catalog # D-4527, Sigma-Aldrich). First, suspended cells were separated using CD45 MACS cell separation magnetic beads (Catalog # 130-052-301, Miltenyi Biotec). Subsequent cell suspensions were sequentially filtered through 100-, 40-, 20 μm filters (Pluriselect, USA). After filtration, cells were in MACS buffer and separated by EpCAM magnetic beads (catalog #130-105-958, Miltenyi Biotec). The resulting CD45(-) EpCAM(+) population was enriched for AEC2 cells followed by flow cytometry and quantitative PCR analysis.

Back skin from CCN1^f/f and CCN1^−/− mice was placed in 10% FBS/DMEM with 2 mg/ml collagenase type II (3 h, 37 °C). Connective tissue was removed and placed into 10% FBS/DMEM. Cell debris was removed by centrifugation (1 min, 500 rpm). The supernatant was filtered (70 μm) and cell pelleted. Cell pellets were cultured in 10% FBS/DMEM (Invitrogen) until 70% confluent, serum-starved overnight in 0.5% FBS/DMEM and treated +/-TGFβ1 (4 ng/mL, 24 h, R and D Systems). Protein was harvested by cell lysis in RIPA buffer; protein (50 μg) was subjected to SDS-PAGE and Western blot analysis with anti-CCN1 (Sc-13,100, 1:800) antibody and HRP-conjugated antibody. Chemiluminescent substrate (Pierce) was added (5 min); X-Ray images were taken. For RNA analysis, cells were similarly treated; however, Trizol (Invitrogen) was used to collect RNA. RNA was used (40 ng/well) for RT-PCR analysis of to detect CCN1 expression using the delta-delta CT method using 18S RNA as an internal control [19 (link),22 ]. Student's t-test was used for statistical analysis.

The cell migration assay was performed using a 24-well transwell chamber with a pore size of 8 µm. In brief, 1×10⁴ cells were seeded in the upper chambers in 200 µl serum-free Dulbecco's modified Eagle's medium (DMEM; Corning Inc., Corning, NY, USA), and 750 µl of 10% FBS-DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) was added into the lower wells. After 24 h at 37°C, cells that had migrated to the bottom of the membrane were fixed and stained with 0.1% crystal violet in methanol in 15 min at room temperature. To quantify the cells, three independent fields per well were photographed under phase contrast microscopy. The number of cells per field were counted and averaged. For the cell invasion assay, the insert of the pore was coated with 50 µl Matrigel (dilution at 1:2; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.