Once the primary murine airway epithelial cells reach 80%confluency, seat cells on a glass slide coated with rat tail collagen. Then, add paraformaldehyde to fix the cells for 12 hours. Wash the slide three times with PBS for five minutes each, and incubate it with 3%bovine serum albumin and 0.1%Triton X-100 in PBS for one hour.
Add anti-pan cytokeratin antibody at a dilution of one to 500 onto the slide, and incubate overnight at four degrees Celsius. The next day, wash the slide three times with PBS solution for five minutes each before incubating with secondary antibody at a dilution of one to 1, 000 for two hours in the dark. After washing the slide with PBS, add DAPI at a dilution of one to 1, 000 and incubate for 15 minutes.
Wash the slide once with PBS for five minutes. Observe the slide under a fluorescence microscope to compare expression patterns to a positive control and a negative control After detachment and centrifugation, re-suspend the P2 phase cells in an appropriate volume of expansion medium. Pipette one milliliter of cell suspension onto the apical chamber of the Transwell polycarbonate membrane insert.
Add 1.5 milliliters of proliferation media to the basal compartment of the Transwell. Incubate the cells at 37 degrees Celsius until confluence is reached. Prepare 50 milliliters of complete differentiation medium using the following components.
Bubble one cigarette through 12.5 milliliters of differentiation medium, and then filter it through a 0.22 micron pour filter to prepare cigarette smoke extract or CSE. To ensure standardization between experiments and batches of CSE, measure the absorbance at 320 nanometers on a spectrophotometer. Next, remove the expansion medium from the apical and basal chamber of Transwells.
Add a differentiation medium containing an appropriate concentration of CSE to stimulate primary murine airway epithelial cells for 28 days. During incubation, wash the apical chamber twice a week with PBS and replace the medium in the basal chamber with freshly prepared differentiation medium containing CSE. Murine airway epithelial cells successfully differentiated at an air liquid interface in 28 days.
The presence of ciliated and goblet cells was demonstrated by immunofluorescence assay of cilia marker, acetylated alpha tubulin, and the goblet cell marker mucin 5AC respectively. Cell viability at 24 hours with varying CSE concentrations demonstrated that epithelial cells declined when the concentration was higher than 6%Cell activity under long-term stimulation showed no significant cell death with 2%4%and 6%CSE concentrations. But changes in transmembrane resistance and exfoliated cells were noted.
In addition, an overall reduction in differentiated cells and ciliated cells was noted when murine airway epithelial cell cultures were chronically exposed to CSE.
