P0884

Mice were randomly assigned into 6 groups: Blank-Control (BC, n = 10), Blank-Stress (BS, n = 10), Propranolol-Control (PC, n = 10), Propranolol-Stress (PS, n = 10), ICI118,551-Control (IC, n = 10) and ICI118,551-Stress (IS, n = 10). Mice in the stress groups experienced physical restraint for 6 hours per day for 35 days commencing 7 days before tumor cell injection. All the mice were sacrificed 28 days after tumor cell injection, and the spleen and liver were harvested and weighed.
For β-adrenergic antagonist studies, (±)-propranolol hydrochloride (a nonselective β-blocker, 10 mg/kg/day, Sigma, P0884) and ICI118,551 (a selective β₂-blocker, 25μM/30μl, Sigma, I127) [17 (link)] were delivered to mice subcutaneously by osmotic minipump (Alzet, Model 1004). Drugs were given for the duration of the experiment commencing 7 days prior to tumor cell injection (Fig 1).

After staining with VSD, cells were qualitatively confirmed to still be spontaneously beating before addition of drugs. Images were captured immediately prior to drug exposure. Medium was then replaced with fresh medium containing either 10⁻⁵ M propranolol (SIGMA, P0884) or 10⁻⁷ M isoproterenol (SIGMA, I6504). We determined the IC₅₀ of propranolol and EC₅₀ of isoproterenol relative to beat rate for these cells was on the order of 10⁻⁶ M and 10⁻⁹ M (S1 Fig). We selected concentrations larger than these to ensure an effective response. Medium without drugs was also replaced as a control. Data was collected 15 min after addition of drugs to ensure complete exposure.