Jc 1 mitoscreen kit

In the early phase of apoptotic death occurring via the intrinsic pathway, there is an increase in mitochondrial membrane permeability, resulting in a decrease in mitochondrial membrane potential (MMP, ΔΨ_m). To detect these changes, the carbocyanine lipophilic cationic fluorochrome JC-1 included in the JC-1 MitoScreen kit (BD Pharmigen, San Diego, CA, USA) and a flow cytometer (BD FACSCanto II) with the appropriate software to analyze the obtained results (FACSDiva; both from BD Biosciences Systems) were used. The entire cell staining and cytometric analysis procedure was performed according to the instructions provided with the kit. MCF-7 and MDA-MB-231 cells were incubated for 24 h (37 °C, 5% CO₂, 90–95% humidity) with cisplatin and compound EDA-71 (concentrations of 1.5 and 3 µM). After the incubation time with the tested compounds, the cells (1 × 10⁶ cells/sample) were washed and resuspended in 0.5 mL of buffer containing 10 µg/mL JC-1 dye. Incubation was carried out for 15 min at room temperature, protected from light. Afterwards, the cells were washed twice with buffer, resuspended in 0.5 mL PBS and immediately analyzed using a flow cytometer (BD FACSCanto II; 10,000 events measured) and FACSDiva software to count the percentage of cells with reduced ΔΨ_m. The equipment was calibrated with BD Cytometer Setup and Tracking Beads (BD Biosciences, San Diego, CA, USA).

Potassium gold(III) chloride, potassium tetrachloropalladate(II), potassium tetrachloroplatinate(II), potassium iodide, acetone, 2-(1-methyl-5-nitroimidazol-2-yl)ethanol, nitric acid(V) silver(I) salt, methanol, diethyl ether, cisplatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), formaldehyde, DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Stock cultures of human breast cancer cell (MCF-7 and MDA-MB-231) and normal human breast epithelial cell (MCF-10A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s minimal essential medium (DMEM), fetal bovine serum (FBS), PBS used in a cell culture, glutamine, penicillin, and streptomycin were obtained from Gibco (San Diego, CA, USA). MEGM Mammary Epithelial Cell Growth Medium BulletKit was purchased from Lonza Bioscience (Basel, Switzerland). FITC Annexin V Apoptosis Detection Kit II, JC-1 MitoScreen Kit and APO-Direct Kit were from BD Pharmigen (San Diego, CA, USA), Autophagy Assay, Red (ImmunoChemistry Technologies, Bloomington, MN, USA), Topoisomerase IIα- PE Conjugate (Cell Signaling Technology, Beverly, MA, USA). Zebrafish were obtained from the Centre for Experimental Medicine of the Medical University of Lublin Bialystok, Poland.