Pictures have been acquired having a QImaging Retiga 2000R camera using the Image Pro Plus 5. 1 software. Quantitative true time PCR and normalization with a number of housekeeping genes RNA extraction and cDNA synthesis, and quantita tive actual time PCR, have been performed as described previously. The analyzed genes and quantitative actual time PCR circumstances are presented in Table 1, except for Star, Hsd3b1, and Cyp11b1. Just after enzyme acti vation, PCR cycles have been performed, 5 sec, denaturation 95 C, five sec, annealing, 20 sec, elongation 72 C, 5 sec, tempera ture of fluorescence intensity reading. Expression stability of three housekeeping genes was assessed depending on the strategy proposed by Vandesompele et al. Then, normalization factors have been calculated from expression levels of the 3 housekeeping genes.
Preparation of fibroblast and epithelial cell enriched cultures from fetal mouse lungs Fetal lungs were removed, rinsed in PBS, cut into pieces of about 2 mm3, and extensively rinsed once again in PBS. Digestion was produced in Hanks buffered saline solu tion containing 0. 25% w v trypsin, 0. 17% w v buy inhibitor collagenase, DNAse I, penicillin streptomycin, and gentamycin at 37 C for 30 minutes beneath agitation. Digestion was stopped by addition of Dulbeccos modified eagles med ium containing 20% v v fetal bovine serum. Residual tissue debris had been discarded by 1 ? g sedimentation and then, supernatants have been centrifuged 7 min at one hundred ? g. Cell pellets were resuspended in DMEM containing 20% v v FBS and penicillin streptomycin. Cells have been incubated for one hour at 37 C with 5% CO2 to enable fibroblast adhesion.
Then, culture media have been removed plus the fibroblast TGX221 adhesion step was repeated twice. The epithelial cell enriched fractions have been collected and filtered through a sterile 45 um cell strainer, centrifuged 7 min at one hundred ? g after which plated in 12 well plates. Cell cultures were kept 12 hours at 37 C beneath 5% CO2 atmosphere. Cell phenotypes have been visually con firmed before harvesting. Epithelial cell enriched cul tures had been then homogenized in TRI Reagent for RNA extraction. Fibroblast enriched cultures were fil tered by way of a sterile 45 um cell strainer just before RNA extraction. 3 separate cell enrichment experiments with two three litters each and every had been accomplished at GD15. 5 and 4 had been performed at GD17. five. Provided the low quantity of epithelial cells recovered at GD15. 5, RNA extracts had been pooled before cDNA preparation. At GD17. 5, adequate epithelial cells for cDNA synthesis had been recovered in 2 out of four experiments. Incubation of lung explants with CRH or ACTH GD 15. five and 17. five fetal lungs were removed, rinsed in PBS, and incubated eight h in DMEM containing penicillin streptomycin and ten 7 M CRH or ten 7 M ACTH at 37 C below gentle agitation. Three and four litters had been analyzed at GD 15.