Veral reports have also indicated that quercetin and isorhamnetin scavenge ROS and inhibit the activation of ERK and MAP kinase in ROS-induced cardiomyopathy [12,13]. In?2013 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Olumacostat glasaretil biological activity Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 2 ofcancer therapy, combining quercetin with doxorubicin augmented the effects of doxorubicin in highly invasive breast cancer cells [14] and can protect cardiomyocytes from doxorubicin-induced toxicity by chelating iron, inducing antioxidant activity, and inhibiting carbonyl reductase [15]. Regarding proteomic analysis, the results also indicated that quercetin could down-regulate Ras GTPase-activating-like proteins and heat shock protein-90 to reduce cell migratory ability and cell survival, respectively, in malignant cancers [16,17]. Although quercetin has been reported to play a role in protecting myocardial cells from ischemia and reperfusion injury, its protective mechanism remains unclear. To investigate the role of quercetin in alleviating doxorubicin-induced cardiotoxicity, we examined the protective ability of quercetin in doxorubicin-treated rat cardiomyocytes by performing cell biological assays, such as cell viability and apoptotic analysis, as well as a quantitative proteomic analysis based on 2D-DIGE and MALDI-TOF MS identification [18].for 10 min prior to incubate with primary antibodies diluted in 2.5 BSA/PBS for 1 h. After PBS washings, samples were incubated with the appropriate fluorescently labeled FCCP site secondary antibodies diluted in 2.5 BSA/PBS for 1 h. Samples were then washed three times with PBS and briefly rinsed with ddH2O twice before applying to Vectashield mounting medium (Vector Lab). Coverslip edges were sealed with nail polish onto glass slides (BDH) and then air-dried in the dark at 4 . For image analysis, cells were visualized using a Zeiss Axiovert Z1 fluorescent microscope (Carl Zeiss Inc., Germany). Identical laser intensities were used to detect the same immunostained proteins to obtain non-saturated images. Images were exported as .tif files using the Zeiss Axioversion 4.0.Flow cytometry analysis for apoptosis detectionMethodsChemicals and reagentsGeneric chemicals were purchased from Sigma-Aldrich (St. Louis, USA), while reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). All primary antibodies were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25768400 purchased from Genetex (Hsinchu, Taiwan) and anti-mouse, and anti-rabbit secondary antibodies were purchased from GE Healthcare (Uppsala, Sweden). All the chemicals and biochemicals used in this study were of analytical grade.Cell lines and cell cultureAnnexin-V/propidium iodide (PI) double assay was performed using the Annexin V, Alexa Fluor?488 Conjugate Detection kit (Life technologies). Following doxorubicin treatment, cells were typsinized from culture dish and washed twice with cold PBS. 1 ?106 cells were resuspended in 500 L binding buffer and stained with 5 L Alexa Fluor 488 conjugated annexin V according to the manufac.Veral reports have also indicated that quercetin and isorhamnetin scavenge ROS and inhibit the activation of ERK and MAP kinase in ROS-induced cardiomyopathy [12,13]. In?2013 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 2 ofcancer therapy, combining quercetin with doxorubicin augmented the effects of doxorubicin in highly invasive breast cancer cells [14] and can protect cardiomyocytes from doxorubicin-induced toxicity by chelating iron, inducing antioxidant activity, and inhibiting carbonyl reductase [15]. Regarding proteomic analysis, the results also indicated that quercetin could down-regulate Ras GTPase-activating-like proteins and heat shock protein-90 to reduce cell migratory ability and cell survival, respectively, in malignant cancers [16,17]. Although quercetin has been reported to play a role in protecting myocardial cells from ischemia and reperfusion injury, its protective mechanism remains unclear. To investigate the role of quercetin in alleviating doxorubicin-induced cardiotoxicity, we examined the protective ability of quercetin in doxorubicin-treated rat cardiomyocytes by performing cell biological assays, such as cell viability and apoptotic analysis, as well as a quantitative proteomic analysis based on 2D-DIGE and MALDI-TOF MS identification [18].for 10 min prior to incubate with primary antibodies diluted in 2.5 BSA/PBS for 1 h. After PBS washings, samples were incubated with the appropriate fluorescently labeled secondary antibodies diluted in 2.5 BSA/PBS for 1 h. Samples were then washed three times with PBS and briefly rinsed with ddH2O twice before applying to Vectashield mounting medium (Vector Lab). Coverslip edges were sealed with nail polish onto glass slides (BDH) and then air-dried in the dark at 4 . For image analysis, cells were visualized using a Zeiss Axiovert Z1 fluorescent microscope (Carl Zeiss Inc., Germany). Identical laser intensities were used to detect the same immunostained proteins to obtain non-saturated images. Images were exported as .tif files using the Zeiss Axioversion 4.0.Flow cytometry analysis for apoptosis detectionMethodsChemicals and reagentsGeneric chemicals were purchased from Sigma-Aldrich (St. Louis, USA), while reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). All primary antibodies were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25768400 purchased from Genetex (Hsinchu, Taiwan) and anti-mouse, and anti-rabbit secondary antibodies were purchased from GE Healthcare (Uppsala, Sweden). All the chemicals and biochemicals used in this study were of analytical grade.Cell lines and cell cultureAnnexin-V/propidium iodide (PI) double assay was performed using the Annexin V, Alexa Fluor?488 Conjugate Detection kit (Life technologies). Following doxorubicin treatment, cells were typsinized from culture dish and washed twice with cold PBS. 1 ?106 cells were resuspended in 500 L binding buffer and stained with 5 L Alexa Fluor 488 conjugated annexin V according to the manufac.