Z1 cell counter

VSMCs and skin fibroblasts were cultured in 12-well plates at 5,000 cells per well in 2 mL DMEM containing 10% FBS. Twenty-four hours after plating, cells were treated with PDGF (20 ng/mL). RU360 (100 nM) was added with PDGF to some wells. After 24–72 h, cells were trypsinized and counted in triplicate using a Beckman Coulter Z1 cell counter.

The BALF was centrifuged to pellet cells, and aliquots of the supernatant were frozen at −80°C until analysis. BALF total protein was measured using a bicinchoninic acid protein assay kit (Pierce). For lung homogenates, perfused lungs were minced, digested with collagenase (150 U/ml; Sigma-Aldrich), and filtered through a 100-µm pore size cell strainer (BD). Red blood cells were removed by hypotonic lysis. The remaining cells were pelleted, resuspended in 1 ml of sterile RPMI 1640, and quantified using an automated cell counter (Z1 cell counter, Beckmann-Coulter). Murine plasma was collected from whole blood mixed with 0.5 M EDTA, and aliquots were frozen at −80°C until analysis. Cytokines were quantified in all samples by ELISA (Mouse IL-6 and KC/CXCL8 DuoSet ELISA kits, R&D Systems).

For short term proliferation assays the cells were UVC irradiated (10 J m⁻²) or not in 60 mm dishes, incubated in fresh media, and then counted in duplicate at various repair time points (0 to 72 hours) using a Beckman Coulter Z1 Cell Counter. The average cell number for each repair time point was divided by the cell number at 0 hour recovery. Cell viability was determined by trypan blue exclusion. Percent viability was calculated as [1.00 – (number of blue cells ÷ number of total cells)] × 100. For long term cell viability assays cells were irradiated with UVC (0, 5 or 10 J m⁻²) or not (untreated) and incubated in fresh media. After 6 hours of recovery the cells were collected by trypsinization and counted, and then subcultured by seeding equal numbers of cells per 10-cm culture dish in duplicate. Following a seven day subculture, the cells were then counted.

A second generation Lentiviral vector system was used. The packaging vectors are psPAX2 and pMDG. The gene expression vectors were transfected with packaging vectors into HEK 293 FT cells to generate lentiviral vectors as previously described (23 (link)). The virus containing cell medium was harvested 48 hours after transfection. The viral vectors were concentrated by ultracentrifuge at 26,000 rpm for 2 hours at 4°C and added to the SW480 cell medium with 8 μg/ml polybrene. Infected cells were subject to the cell proliferation assay. Cells (150,000) were seeded in 60 mm plates on day 0 and then trypsinized and counted after 24, 48, 72 and 96 hours by a Coulter Z1 cell counter as previously described (24 (link)). Experiments were run in triplicate.