Turer’s instructions. 1 L 100 g/mL propidium iodide (PI) was mixed

Turer’s instructions. 1 L 100 g/mL propidium iodide (PI) was mixed gently to cells for 15 min at room temperature in the dark. After incubation period, samples were subjected to FCM analysis in 1 h. using BD Accuri C6 Flow Cytometry (BD Biosciences, San Jose, CA). The data were analyzed using Accuri CFlow@ and CFlow Plus analysis software (BD Biosciences).Immunoblotting analysisViability ( )The rat cardiomyocyte cell line H9C2 was purchased from American Type Culture Collection (ATCC) (SetmelanotideMedChemExpress BIM-22493 Manassas, VA) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 (v/v) FCS, L-glutamine (2 mM), streptomycin (100 g/mL) and penicillin (100 IU/mL) (all from Gibco-Invitrogen Corp., UK). Cells were incubated in a humidified incubator at 37 and 5 CO2. and passaged at 80-90 confluence by trypsinization according to standard procedures.MTT cell viability assayImmunoblotting analysis was used to validate PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 the differential abundance of mass spectrometry identified proteins. The detailed experimental procedures were described in100 9060 50The detailed MTT experimental procedure has been described in our previous study [19].Immunofluorescence20 10 0 0 0.2 0.4 0.6 0.8Cells were plated onto coverslips (VWR international) for overnight incubation and subsequently fixed with PBS containing 4 (v/v) paraformaldehyde for 25 min. After washing three times in PBS, samples were permeabilized in PBS containing 0.2 (v/v) Triton X-100 for 10 min. and then rinsed and blocked in PBS containing 5 (w/v) BSADox conc. ( )Figure 1 Effect of doxorubicin treatment on H9C2 cell viability. H9C2 cells were treated with indicated concentrations of doxorubicin from 3 independent experiments. Cell viability was determined by MTT assay after 24 h exposure of doxorubicin. Each data point indicates mean ?SD of triplicate values.Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 3 ofA***2.50 2.*** *** ***1.52 1.00 2.06 2.19 1.CDoxQue Caspase-3 Caspase-9 GAPDHRecovery Index1.1.0.50 0.00 0 50 100 150Quercetin ( )BFL2-H / Propidium idideControlDoxQue + Dox* *Late apoptosis ( )40 35 30 25 20 155 0 Ctrl Dox Dox+QueFL1-H / Annexin VDControlDoxQue + Dox505050202020Figure 2 (See legend on next page.)Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 4 of(See figure on previous page.) Figure 2 Effects of quercetin on doxorubicin-induced changes of cell viability, cell apoptosis and cell morphology in H9C2 cells. (A) MTT-based viability assays were performed on H9C2 cell cultures following BUdR price treatments with different concentrations of quercetin (50 M, 100 M, 150 M and 200 M) or left untreated. Values were normalized against untreated samples and were the average of 4 independent measurements +/- the standard deviation. The statistic analysis was performed with two group paired Student t-test. (B) Typical dot plot diagrams detected annexin V-FITC and PI staining represent untreated, doxorubicin-treated, and quercetin-pretreated followed by doxorubicin reated cells. The x-axis and y-axis stand for the intensity of annexin V-FITC and PI, respectively. The lower left area of presented background staining by annexin V-FITC and PI in normal cells, and apoptotic signals located in the right area. This figure is representative of 4 replicates. The statistic analysis of the replicates was listed in right panel. (C) The levels of caspase 3 and caspase 9 in H9C2 cells were detected by.Turer’s instructions. 1 L 100 g/mL propidium iodide (PI) was mixed gently to cells for 15 min at room temperature in the dark. After incubation period, samples were subjected to FCM analysis in 1 h. using BD Accuri C6 Flow Cytometry (BD Biosciences, San Jose, CA). The data were analyzed using Accuri CFlow@ and CFlow Plus analysis software (BD Biosciences).Immunoblotting analysisViability ( )The rat cardiomyocyte cell line H9C2 was purchased from American Type Culture Collection (ATCC) (Manassas, VA) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 (v/v) FCS, L-glutamine (2 mM), streptomycin (100 g/mL) and penicillin (100 IU/mL) (all from Gibco-Invitrogen Corp., UK). Cells were incubated in a humidified incubator at 37 and 5 CO2. and passaged at 80-90 confluence by trypsinization according to standard procedures.MTT cell viability assayImmunoblotting analysis was used to validate PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 the differential abundance of mass spectrometry identified proteins. The detailed experimental procedures were described in100 9060 50The detailed MTT experimental procedure has been described in our previous study [19].Immunofluorescence20 10 0 0 0.2 0.4 0.6 0.8Cells were plated onto coverslips (VWR international) for overnight incubation and subsequently fixed with PBS containing 4 (v/v) paraformaldehyde for 25 min. After washing three times in PBS, samples were permeabilized in PBS containing 0.2 (v/v) Triton X-100 for 10 min. and then rinsed and blocked in PBS containing 5 (w/v) BSADox conc. ( )Figure 1 Effect of doxorubicin treatment on H9C2 cell viability. H9C2 cells were treated with indicated concentrations of doxorubicin from 3 independent experiments. Cell viability was determined by MTT assay after 24 h exposure of doxorubicin. Each data point indicates mean ?SD of triplicate values.Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 3 ofA***2.50 2.*** *** ***1.52 1.00 2.06 2.19 1.CDoxQue Caspase-3 Caspase-9 GAPDHRecovery Index1.1.0.50 0.00 0 50 100 150Quercetin ( )BFL2-H / Propidium idideControlDoxQue + Dox* *Late apoptosis ( )40 35 30 25 20 155 0 Ctrl Dox Dox+QueFL1-H / Annexin VDControlDoxQue + Dox505050202020Figure 2 (See legend on next page.)Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 4 of(See figure on previous page.) Figure 2 Effects of quercetin on doxorubicin-induced changes of cell viability, cell apoptosis and cell morphology in H9C2 cells. (A) MTT-based viability assays were performed on H9C2 cell cultures following treatments with different concentrations of quercetin (50 M, 100 M, 150 M and 200 M) or left untreated. Values were normalized against untreated samples and were the average of 4 independent measurements +/- the standard deviation. The statistic analysis was performed with two group paired Student t-test. (B) Typical dot plot diagrams detected annexin V-FITC and PI staining represent untreated, doxorubicin-treated, and quercetin-pretreated followed by doxorubicin reated cells. The x-axis and y-axis stand for the intensity of annexin V-FITC and PI, respectively. The lower left area of presented background staining by annexin V-FITC and PI in normal cells, and apoptotic signals located in the right area. This figure is representative of 4 replicates. The statistic analysis of the replicates was listed in right panel. (C) The levels of caspase 3 and caspase 9 in H9C2 cells were detected by.