User:Abhishektiwari/Notebook/Hormone Dynamics of HAC15 Cells/2010/01/21

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HAC Media Information

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).


HAC15 human adrenal carcinoma cells were plated in DMEM/F12 with L-glutamine and HEPES (Invitrogen) media with 10% cosmic calf serum (Fisher Scientific, Pittsburgh, PA), 1 µg/ml gentamicin (Invitrogen), and 1% insulin/transferrin/selenium premix (BD Bioscience) at 400,000 cells/well in 24-well tissue culture plates. Cells were allowed to adhere to plates and grow for 1 d, and media were replaced with a low serum media (0.1% cosmic calf serum).


H295R human adrenal carcinoma cells were plated in DMEM/F12 (Invitrogen, San Diego, CA) media with 2.5% {nu} serum, and 1% insulin/transferrin/selenium premix (BD Biosciences, San Jose, CA) at 500,000 cells/well in 24-well tissue culture plates. Cells were allowed to adhere to plates and grow confluent for 1–2 d and then treated with vehicle dimethyl sulfoxide, angiotensin II (Sigma-Aldrich, St. Louis, MO), or test compound. At 24 and 48 h time points, 500 µl media were removed from each well and frozen at –20 C until needed for aldosterone and cortisol assays. Cells were lysed in buffer RLT (QIAGEN, Valencia, CA). Lysate was frozen at –80 C until RNA extraction.

Y-1 mouse adrenal cells

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.

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.


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