Steroids Effect on HUVEC Response to LPS or Cytokines

Pooled first passage cryopreserved, human umbilical vein endothelial cells (HUVEC) were obtained from Cascade Biologics (Portland, OR), thawed, and cultured according to the supplier’s recommendations in Medium 200. After thawing, cells were seeded onto 0.2% gelatin coated tissue culture plates and cultivated under 95% air, 5% CO2 at 37 oC. Confluent cells were cultured in steroids-depleted medium for 96 hrs before the addition of 17β-estradiol (17βE) or hydrocortisone (H) to 1 µM for 5 hrs. For Lipopolysaccharide (LPS) or cytokine mix (CM)-treatments, cells or pretreated with vehicle (0.1% ethanol), 1µM 17βE, or H for 1 hr were exposed to 100 ng/ml LPS or a mixture of proinflammatory cytokines (CM, 500 U/ml IL-1b, 2500 U/ml TNF-α, and 1250 U/ml IFN-γ) for 4 hours. All treatments were done in quadruplicates. At the end of treatment, total cellular RNA was isolated. Keywords: parallel sample