H9C2 cells can be seeded in a 6-well plate at a density of 1.0 × 105 cells/well and maintained for 24 h in DMEM medium containing 10% FBS. Fas siRNA (1.0 μg) is complexed with polymer at different weight ratios and incubated for 30 min. The cells can be treated with polymer:Fas siRNA polyplexes for 4 h in serum-free media and then further incubate for 24 h in fresh DMEM medium containing 10% FBS. For hypoxic conditions, the cells transfected with Fas siRNA polyplexes were incubated in a hypoxia chamber (BD GasPak EZ system, San Diego, CA), which catalytically reduces oxygen to undetectable levels within 150 minThe cells, NIH 3T3 and H9C2, can be seeded in 24-well plates at a density of 5.0 × 104 cells/well and incubate for 24 h in DMEM medium containing 10% FBS at 37 °C.
For short-term studies: ACCT medium: M199 supplemented with 2 g/L fatty acid-free BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurin and 10 nM triiodothyronin
For longer-term studies we use M199 supplemented with 20% FBS, 20 mM creatine, 2 mM cytosine-beta-D-arabinofuranoside (a.k.a. araC, prevents growth of non-myocyte cells) and 100 nM 9-cis retinoid acid. This medium induces extensive remodeling and hypertrophy of the cardiac myocytes but with inclusion of retinoid acid the cells retain a fairly well differentiated phenotype (see Montessuit C, et al. Basic Res. Cardiol. 2006 101: 27-35).
thanks, used that with rat cells fine but for some reason we think with rabbit cells, the tubules/sarcomere/overall morphology is best preserved in the mystery ingredients of PC1
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