All reagents and media were warmed to 37°C prior to use. The amniotic membrane was processed as described previously [6 , 12 (link), 14 (link)] Briefly, the amnion membrane was manually peeled from normal, term, not in labor caesarean section placentas, rinsed in saline and transferred to a petri dish containing Hanks Balanced Salt Solution (HBSS; Mediatech Inc., Manassas, VA). After cutting the amnion into 2 cm x 2 cm pieces, they were digested twice in 0.25% trypsin and 0.125% Collagenase A (Sigma–Aldrich, St. Louis, MO) in HBSS for 35 minutes at 37°C. After each digestion, the tissue was filtered through a 70 μm cell strainer (Thermo Fisher Scientific, Waltham, MA) and trypsin was inactivated using complete Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 media (DMEM/F12; Mediatech Inc.) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich), 10% Penicillin/Streptomycin (Mediatech Inc.) and 100 μg/mL epidermal growth factor (EGF; Sigma-Aldrich). The collected filtrate was centrifuged for 10 minutes at 3000 RPM and the pellet was resuspended in 3.0 mL complete DMEM/F12. Once cells were counted, approximately 3–5 million cells per flask were cultured in T75 flasks containing complete DMEM/F12 media at 37°C, 5% CO2, and 95% air humidity to 70–80% confluence.
To ensure the purity of our primary AEC cultures, immunofluorescent staining was performed. Cells were seeded on glass coverslips at a density of 30,000 cells per slip and incubated overnight. Cells were fixed with 4% paraformaldehyde (PFA), permeablized with 0.5% Triton X and blocked with 3% BSA in PBS prior to incubation with Cytokeratin 18 (Abcam, Cambridge, United Kingdom) primary antibody diluted 1:300 in 3% BSA overnight at 4°C. After washing with PBS, slides were incubated Alexa Fluor conjugated secondary antibodies (Life Technologies, Carlsbad, CA) diluted 1:400 in PBS for 1 hour in the dark. Slides were washed with PBS then treated with NucBlue® Live ReadyProbes® Reagent (Life Technologies) then mounted using Mowiol 4–88 mounting medium (Sigma-Aldrich). Images were captured using LSM 510 Meta UV confocal microscope (63x) (Zeiss, Germany).
To ensure the purity of our primary AEC cultures, immunofluorescent staining was performed. Cells were seeded on glass coverslips at a density of 30,000 cells per slip and incubated overnight. Cells were fixed with 4% paraformaldehyde (PFA), permeablized with 0.5% Triton X and blocked with 3% BSA in PBS prior to incubation with Cytokeratin 18 (Abcam, Cambridge, United Kingdom) primary antibody diluted 1:300 in 3% BSA overnight at 4°C. After washing with PBS, slides were incubated Alexa Fluor conjugated secondary antibodies (Life Technologies, Carlsbad, CA) diluted 1:400 in PBS for 1 hour in the dark. Slides were washed with PBS then treated with NucBlue® Live ReadyProbes® Reagent (Life Technologies) then mounted using Mowiol 4–88 mounting medium (Sigma-Aldrich). Images were captured using LSM 510 Meta UV confocal microscope (63x) (Zeiss, Germany).
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