Page 57 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

bovine serum (Cambrex, Verviers, Belgium) in 175 cm2 flasks (Fisher Scientific B.V., Landsmeer, The Netherlands).
Labeling of cells with SPIO was performed at 80–90% confluence using Endorem (Guerbet S.A., Paris, France) and lipofectamine 2000 (Invitrogen, Breda, The Netherlands). A labeling stock solution of 215 ug Fe ml-1, for a 175 cm2 flask, was prepared as follows: 25 ul Endorem was added to 625 ul Opti-MEM (Invitrogen, Breda, The Netherlands) and 25 ul of lipofectamine was added to 625 ul Opti-MEM. After 5 min the two solutions were mixed together and the resulting suspension was incubated at room temperature for 20 min. After washing the cells with phosphate-buffered saline (PBS; Invitrogen, Breda, The Netherlands), the culture medium was replaced by advanced RPMI medium with 1% (v/v) penicillin–streptomycin and 233 ug (1082 ul) of iron was added to the cells, which were then incubated for 24 h at 37oC–5% CO2. Before further use of labeled cells, the monolayer cultures were rinsed three times with PBS.
Phantom preparation
For the MR measurements, phantoms were prepared consisting of six 0.5 ml Eppendorf tubes as sample holders. The sample holders were placed in 3 cm diameter dishes filled with water to avoid the proximity of an air interface (Fig. 2a and b). The samples were prepared with 0.3% agar (Becton Dickinson, Alphen aan de Rijn, The Netherlands) in order to avoid sedimentation. The cells used for the samples were fixed with 4% formaldehyde (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 10 min at room temperature.
Incorporation of iron, localization of iron complexes and label retention
The used agar samples were dried for 72 h at 60 oC. Subsequently they were digested in 40 ul of a 3:1 mixture of ultra-pure perchloric acid (EM Science, Gibbstown, NJ, USA) and ultrapure nitric acid (JT Baker, Deventer, The Netherlands) at 60 oC for 6 h. To the digested substance 4 ml MiliQ was added and the amount of iron was determined with a Perkin Elmer Optical Emission Optima 4300 DV Spectrometer (Perkin Elmer Instruments, Norwalk, NC, USA) at 259 nm. The experiments were performed three times with triplicate samples. To assess the presence and distribution of the iron complexes, cytospin slides were prepared from labeled cells. After the slides were dried by air the cells were fixed in absolute methanol (Sigma-Aldrich, Zwijndrecht,
Quantification of iron labeled cell
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