User:Abhishektiwari/Notebook/Hormone Dynamics of HAC15 Cells/2010/01/21
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HAC Media Information
<xfeeds pubget="Y">doi:10.1210/jc.2008-0903</xfeeds> Primary carcinoma cells were isolated after the tumor was minced into small pieces and incubated in Dulbecco’s Modified Eagle/F12 (Invitrogen, Carlsbad, CA) containing 0.1% collagenase (Roche, Indianapolis, IN). Digestion and mechanical dispersion were carried out for 1.5 h at 37 C, with a final digestion of the dispersed cells in medium containing 0.01% deoxyribonuclease I. After isolation, cells were frozen in DME/F12 medium, 50% Nu Serum (BD Biosciences, Franklin Lakes, NJ), and 10% dimethyl sulfoxide (Sigma, St. Louis, MO). The frozen aliquots were later suspended in growth media consisting of DME/F12 medium supplemented with 10% cosmic calf serum (HyClone, Logan, UT), antibiotics and 1% insulin/transferrin/selenium Premix (BD Biosciences) and plated at cloning density. After 3 wk, clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, HAC clone 15 (HAC15), was used for this study. H295R cells, grown under similar conditions were used for comparison studies. For experiments, cells were plated onto 12 dishes (400,000 cells/well) in growth medium. Cells were treated in low serum medium (0.1% cosmic calf serum) with Ang II (10 nM; Sigma), forskolin (10 µM; Sigma), or ACTH (10 nM; Organon, Bedford, OH).
Y-1 mouse adrenal cells
<xfeeds pubget="Y">doi:10.1210/en.2009-0551</xfeeds> Y-1 mouse adrenocortical cells purchased from American Type Culture Collection (Manassas, VA) were cultured in Ham’s F12K culture medium containing 2 mM L-glutamine, supplemented with 1.5 g/liter sodium bicarbonate, 15% horse serum, 2.5% fetal bovine serum, and 1% penicillin-streptomycin mix.
<xfeeds pubget="Y">doi:10.1016/j.bbrc.2009.10.122</xfeeds> The mouse adrenocortical cell line Y-1 was maintained in F-12K (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 15% horse serum, 2.5% fetal bovine serum (FBS), 100 lU/ml penicillin and 100 μg/ml streptomycin in a 37 °C, 5% CO2 atmosphere. Y-1 cells were cultured in 24-well plates at a density of 2 × 105 cells/well.