Page 47 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

5.4. Incorporation of iron
5.4.1. Inductively coupled plasma–optical emission spectrometry
Cell pellets of unlabeled and labeled cells were dried for 72 h at 60°C. Subsequently they were digested in 40 ml of a 3:1 mixture of ultra-pure perchloric acid (EM Science, Gibbstown, NJ, USA) and ultra-pure nitric acid (JT Baker, Deventer, The Netherlands) at 60°C for 6 h. The standard line with different dilutions of Endorem or MPIO underwent the same procedure as the cells. 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 at 259 nm. The experiments were performed three times with triplicate samples. Uptake efficiency was calculated as the amount of iron measured in the labeled cells divided by the total amount of iron added for labeling multiplied by 100%.
5.4.2. Fluorescence activated cell sorting
The MPIO were tagged with Dragon Green, which is fluorescent. To analyze the amount of MPIO taken up by the HUVEC and to assess the effects of MPIO on morphological characteristics of the cell and cell survival, a FACS (FacsCanto, Becton Dickinson, Alphen a/d Rijn, The Netherlands) analysis was performed. After incubation with MPIO all the cells were collected and resuspended in FACS buffer (0.25% BSA, 0.5 mM EDTA, 0.05% NaN3), and 1% v/v propidium iodide (all from Sigma-Aldrich, Zwijndrecht, The Netherlands) was added to determine the amount of dead cells in the suspension. The wavelength used was 488 nm. Cell size was estimated using the forward scatter parameter and the side scatter parameter was used to determine the cytoplasmic granularity.
5.5. Localization of iron complexes and label retention
For assessing the localization of the iron complexes within the cell, cytospin slides were prepared from labeled cells. A cell suspension of 2 x 105 cells/ml was made and 150 ml was loaded in each cuvette. The cells were centrifuged (7 min, 800 rpm; Shandon Inc.) onto the slide. After the slides were dried by air the cells were fixed in methanol absolute (Sigma-Aldrich, Zwijndrecht, The Netherlands) and Prussian blue staining (Sigma-Aldrich, Zwijndrecht, The Netherlands) was performed. The slides were covered with a cover slip and imaged with an Axiovert S100 microscope (Zeiss, Oberkochen, Germany).
Influence of SPIO cell labeling protocol
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