Cell viability is determined using a Cell Counting Kit-8 assay. Briefly, 100 μL Raji cells (5×10⁴/mL) are seeded in triplicate in a 96-well plate and treated with various concentrations of ouabain (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 nM) for 48 h. Following the 48-h treatment, 10 μL CCK-8 reagent is added to each well, and the cells are incubated for an additional 3 h at 37°C. Optical density (OD) values at 450 nm are subsequently measured, and each ouabain concentration is assessed in triplicate. Raji cells cultured in medium without drug served as controls. Cell viability is calculated according to the following formula: Inhibition rate (%)=[1 ? (OD_450(sample) ? OD_450(blank))/(OD_450(control) ? OD_450(blank))] × 100.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
A total of 8 mice are used in the present study, 4 in the control group and 4 in the ouabain-treated group. Animals are maintained under standard laboratory conditions, with room temperature controlled (22°C), and subjected to 12 h light-dark cycles with ad libitum access to food and water. At 24 h subsequent to the intraperitoneal injection with 300 μg/kg of ouabain or PBS, the Swiss mice are sacrificed by barbiturate overdose (86 mg/kg intraperitoneal injection of pentobarbital). The mesenteric lymph nodes and thymi are immediately removed and softly dissociated. The remaining cells are washed in PBS and centrifuged at 200 × g. The pellet is suspended in ice-cold RPMI-1640 culture medium supplemented with 10% FBS until required for the activity assays.
Rat^[3]
Male Wistar rats are treated with daily intraperitoneal injections of 30 μg/kg of ouabain or its vehicle, phosphate-buffered saline (PBS). A total of 20 rats are used, 12 for acute treatment (n=6 rats/group in ouabain and control groups) and 8 for chronic treatment (n=4 rats/group in ouabain and control groups). Animals are maintained under standard laboratory conditions, with room temperature controlled (22°C), and subjected to 12 h light-dark cycles with ad libitum access to food and water. Prior to the first injection at 24 h and 7 and 14 days subsequent to the injection, the rats have their blood pressure measured by a computerized tail-cuff method. The animals are sacrificed by barbiturate overdose (86 mg/kg intraperitoneal injection of pentobarbital) after 24 h (acute treatment) or 14 days (chronic treatment) of ouabain injections, and the mesenteric lymph nodes, thymi and blood are collected. Full excisions of thymi and partial excisions of mesentheric lymph nodes are performed, while blood samples are collected by caudal venous puncture prior to animals sacrifice.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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