Materials and Methods

Synthesis and characterization of Co-SPIONs. Co-SPIONs were synthesized in the thermostated glass reactor by Massart’s co-precipitation method (Massart, 1981) from the alkaline solutions of Co(II) and Fe(III) metal salts at 70 °C for 5 h. All reagents in synthesis procedure were at least of analytical grade and were used without any further purification, except for NaOH, which was purified by preparation of a saturated solution resulting in the crystallization of other sodium salts. CoCl2, Fe2(SO4)3 and citric acid were purchased from Aldrich Chemicals Inc. Ultrapure water was used throughout all experiments. For preparation of the working solutions 0.12 mol/L CoCl2, 0.06 mol/L Fe2(SO4)3, 5.0 mol/L NaOH and 0.3 mol/L citric acid solutions were prepared and deoxygenated with argon before mixing. The molar ratio of cobalt(II) and iron(III) salts in the reactor was 1:1.2 at their total concentration - 40 mmol. The pH of solutions was maintained at 11.5. The required quantity of 5.0 mol/L NaOH was determined by additional blank experiment. In the subsequent experiment, the estimated amount was added to the reactor, containing all other components, in several seconds under vigorous stirring. The synthesis in the thermostated reactor was conducted under a continuous argon gas bubbling. The crude products were centrifuged at 7500 rpm for 5 min and rinsed several times. The supernatants obtained from the last three centrifugations were combined, neutralized by addition of citric acid solution up to pH 6.0 and used as a stable ferrofluid within the following week. The composition of the synthesized products was investigated by energy dispersive X-ray spectroscopy and following nanoparticle dissolution in HCl (1:1) solution by inductively p...

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... 7-aminoactinomycin D (BD Biosciences, USA) staining.
Magnetic-activated cell sorting. Magnetic-activated cell sorting (MACS) was used to separate magnetically labeled and unlabeled cells after 48 hours of incubation with Co-SPIONs. A single-cell suspension of treated cells was passed through a LS Column, placed in a MidiMACS Separator (Miltenyi Biotec, Germany). Magnetically-unlabeled fraction flew freely through the column. Magnetically labeled fraction was collected by removing LS Column out of magnetic field and eluting attached cells with PBS. Both fractions were examined under the microscope and cell counter for presence of cells.
Statistical analysis. Triplicate measurements of each sample were performed in three independent experiments. Statistical differences between the groups were analysed by Student’s t-test. A value of p<0.05 was considered significant.