The purpose of this study was to evaluate the cytotoxicity of human multiple myeloma cells (RPMI-8226) treated with graphene oxide (GO), doxorubicin (DOX), and GO loaded with DOX (GO/DOX). medium. Analysis of cell viability Cell buy 313967-18-9 viability was assessed using the CCK-8 assay in a microplate reader. RPMI-8226 cells were seeded in Mouse monoclonal to EphA5 96-well microplates (Corning Technologies, Corning, NY, USA) at a density of 1.5105 cells/mL in 100 L RPMI-1640 medium containing 10% FBS for 24 hours. Cells were then cultured in medium with various concentrations of GO for 24 hours. Control cells did not receive any GO treatment. Three replicate wells were used for the control and test concentrations. Ten microliters of CCK-8 was added to each well, and the microplate was incubated at 37C for 2 hours in a 5% CO2 humidified incubator. The absorbance was then measured at 450 nm using a microplate reader (Spectrafluor; Tecan, M?nnedorf, Switzerland). Cell viability was expressed as a percentage of the buy 313967-18-9 control buy 313967-18-9 cell culture value. A control was performed in parallel to monitor the influence of RPMI-1640 medium on the assays. The cell viability was calculated as follows: for 5 minutes at room temperature, washed twice with ice-cold phosphate buffered saline (PBS), and fixed with 70% ethanol at 4C overnight. The fixed cells were suspended in PBS and further treated with propidium iodide (PI) for 30 minutes at 37C in the dark. The cells were then centrifuged at 1, 000for 5 minutes at room temperature and washing twice with ice-cold PBS. The buy 313967-18-9 cell density was calculated and the cells resuspended in 1 annexin-binding buffer to obtain a final density of 1106 cells/mL. One-hundred microliters of the cell solution was placed into 1.5 mL Eppendorf tubes and 5 L annexin V-fluorescein isothiocyanate (FITC) and 1 L PI (100 g/mL) working solution added. The RPMI-8226 cells were incubated at room temperature for 15 minutes. After incubation, 400 L of 1 annexin-binding buffer was added, gently mixed, and the samples kept on ice. The DNA (deoxyribonucleic acid) content of the cells was analyzed by flow cytometry (Cytomic? FC500). All testing was required to be completed within an hour. Statistical buy 313967-18-9 analysis Three replicates of each treatment concentration were performed for each analysis. Values were expressed as mean standard deviation of three independent experiments. Comparisons between two groups were analyzed using one-way analysis of variance, with P<0.05 taken as statistically significant. Results Effects of GO on cell viability RPMI-8226 cells were treated with different concentrations of GO for 24 hours, and the effect of GO on cell viability assessed using the CCK-8 assay. Treated cells showed a GO dose-dependent decrease in cell viability (Figure 1). The control had 100% viable cells. Increasing GO concentration from 10 to 100 mg/L decreased cell viability from 95.6% to 79.6%, respectively. These results suggest that GO caused low cytotoxicity in RPMI-8226 cells. Figure 1 The effect of GO (10, 25, 50, and 100 mg/L; size <100 nm) on cell viability of RPMI-8226 cells for 24 hours. Effects of DOX and GO/DOX on MM cell morphology Morphological studies were performed to identify the mode of cell death in MM cells. The morphology of untreated, GO-treated, DOX-treated, and GO/DOX-treated cells was monitored by optical microscope at 24 hours. Untreated cells were round and large, with a bright cytoplasm and good refraction (Figure 2A). GO-treated cells were round with evidence of cell shrinkage and a translucent cytoplasm (Figure 2B). DOX-treated cells exhibited typical apoptotic features, such as membrane blebbing and cell shrinkage (Figure 2C). GO/DOX-treated cells showed significant morphological changes, including cell shrinkage (Figure 2D). These results suggest that DOX and GO/DOX might have induced apoptosis in RPMI-8226 cells but not GO. Figure 2 Morphology of RPMI-8226 cells treated with different drugs. Cells were treated for 24 hours with control (0 mg/L) (A), GO (50 mg/L).