I am using healthy control lung fibroblasts in a 96 hour experiment with two drugs (nintedanib and Pirfenidone) and vehicle controls for both. I have both hypoxia (approx 1% O2) and normoxia conditions and would expect to see an increase in proliferation for hypoxic VC after 96 hours - however this is not the case. I have produced a result in which hypoxia did increase proliferation versus normoxia VC but over a 72 hour incubation. The key differences between these experiments is that the 72 hour experiment allows for an overnight adhesion to plate step following seeding before treatments are added while the 96 hour experiment dictates that the treatment is added 6 hours after seeding the plates (due to time constraints). Does anyone have any experience with this type of experiment and come into similar issues?
Dear Robert!
Please you look in the article:
Article Hypoxia induces pulmonary fibroblast proliferation through N...
Primary human pulmonary fibroblasts (HPFs)
Hypoxia exposure
Cells were exposed to normoxia (21% O2) in a normal CO2 cell culture incubator or hypoxia (5% or 1% O2) in a hypoxia chamber (Billups-rothenburg, Del Mar, CA). Sterile water was placed inside the chamber to maintain moist conditions. Hypoxic conditions were created by filling the chamber with 5% or 1% O2, 5% CO2 and balanced N2. The gas was passed through the chamber at 1–2 psi for 3 min, and the chamber was sealed and placed in a 37 °C cell culture incubator. The oxygen concentration in the chamber was monitored using an oxygen sensor (OxyCheq, Marianna, FL). Every 2 days, media were replaced with fresh media that were degassed using a SpeedVac Concentrator system (Thermo Fisher Scientific, Waltham, MA).
Figure 1
Hypoxia induces pulmonary fibroblast proliferation. Normal human pulmonary fibroblasts (HPFs) and LL29 IPF fibroblasts were exposed to normoxia (21% O2) or hypoxia (5% or 1% O2) for 2–6 days.
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