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. 2017 Mar 10;22(3):437. doi: 10.3390/molecules22030437

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© 2017 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Bufalin induced reactive oxygen species and Ca²⁺ production and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in U-2 OS cells. Cells (1 × 10⁵ cells/well) were treated with bufalin (200 nM) for various time periods. Cells were isolated and were re-suspended in 500 μL of DCFH-DA (2′,7′-Dichlorofluorescin diacetate) (10 μM) for further ROS (Reactive oxygen species) (H₂O₂) (A: short time, B: long time), (C), re-suspended in 500 μL of Fluo-3/AM (2.5 μg/mL) for further intracellular Ca²⁺ concentrations, and (D) re-suspended in 500 μL of DiOC₆ (4 μmol/L) for 30 min for further levels of ΔΨ_m measurement, as described in Materials and Methods. The results are shown as a mean ± SD (n = 3); * p < 0.05, significant difference between bufalin-treated groups and the control as analyzed by Student’s t test.

Bufalin induced reactive oxygen species and Ca²⁺ production and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in U-2 OS cells. Cells (1 × 10⁵ cells/well) were treated with bufalin (200 nM) for various time periods. Cells were isolated and were re-suspended in 500 μL of DCFH-DA (2′,7′-Dichlorofluorescin diacetate) (10 μM) for further ROS (Reactive oxygen species) (H₂O₂) (A: short time, B: long time), (C), re-suspended in 500 μL of Fluo-3/AM (2.5 μg/mL) for further intracellular Ca²⁺ concentrations, and (D) re-suspended in 500 μL of DiOC₆ (4 μmol/L) for 30 min for further levels of ΔΨ_m measurement, as described in Materials and Methods. The results are shown as a mean ± SD (n = 3); * p < 0.05, significant difference between bufalin-treated groups and the control as analyzed by Student’s t test.