4) twice. After fixing and sectioning, cells were dehydrated via ethanol and stained with 5% uranyl acetate for 30 min followed by Reynold’s lead citrate incubation [27]. Stained cells were examined under JEOL 2100F transmission electron microscope. The presence of INPs and CSO-INPs in mitochondria surface and matrix was further confirmed by the TEM-EDS elemental analysis (TEM, JEOL 2100F). For apoptosis analysis HeLa, A549 and Hek293 cells were seeded at the density of 1 × 105 cells/well and incubated at 37 °C for 24 h. Cells were treated

with 4 μg/μl of INPs and CSO-INPs respectively for 48 h. Cells were trypsinized using 1× trypsin–EDTA and LGK-974 solubility dmso pooled in 1.5 ml tube, washed with 1× PBS buffer. Cells were resuspended Epigenetics Compound Library in 500 μl of 1× Annexin binding buffer [10× buffer composition: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2], 1 μl of Annexin V-FITC reagent used from stock (final

concentration 1 μg/ml). Stained samples were gently mixed and incubated at 37 °C for 10–20 min in dark [28]. FL1 channel was applied for detecting Annexin V-FITC staining through flow cytometry (BD Biosciences) with excitation wavelength 488 nm. Fluorescence spectra were analyzed by FCS 4 Express Flow Cytometry software. Mitochondrial membrane potential was analyzed by JC-1 probe (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-benzimidazolylcarbocyanine iodide) staining [29] and [30]. HeLa, A549 and Hek293 cells (1 × 105 cells/well) were harvested after 48 h exposure of 4 μg/μl iron oxide nanoparticles and chitosan oligosaccharide coated iron oxide nanoparticles (CSO-INPs) and centrifuged at 400 × g for 5 min. Cell pellet was resuspended in 0.5 ml of JC-1 solution

(10 μg/ml) for 10 min. Cells were washed with 1× PBS buffer. Mitochondrial depolarization is identified by reduction of the red/green fluorescence ratio. Green fluorescence DCLK1 (monomers) was observed through FL1 channel with almost 10,000 events of each sample using flow cytometry (BD Biosciences) with excitation at 488 nm wavelength. Fluorescence spectra were analyzed by FCS 4 Express Flow Cytometry software. Analysis of ROS production was carried out using the method reported by Mancini et al. [31], with slight modification. HeLa, A549 and Hek293 cells (1 × 105 cells/well) were seeded and incubated at 37 °C in CO2 incubator for 24 h. The cells were treated with 4 μg/μl iron oxide nanoparticles (INPs) and chitosan oligosaccharide coated iron oxide nanoparticles (CSO-INPs) respectively for 48 h. Cells were trypsinized with 1× trypsin–EDTA, and centrifuged at 1000 rpm for 5 min. Cells were washed twice with 1× PBS buffer (pH 7.4) followed by 1 h incubation in DCFH-DA (10 μmol/l) in FBS-free DMEM medium. Cells were resuspended in 1× PBS buffer, subjected to flow cytometry analysis. Finally, fluorescence spectrum was measured by flow cytometry (BD Biosciences) at 488 nm excitation and emission at 530 nm wavelength for DCFDA with 10,000 events of each sample.