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Figure 1. Superparamagnetic iron oxide particle labeling protocol optimization of BV-2 cells. DTR/eGFP-transduced and control cells were labeled with 10, 25 and 50 µgFe/ml of ferucarbotran (Resovist(TM)), and 25, 50 and 100 µgFe/ml of ferumoxide (Endorem(TM)).
(A) In total, 60,000 cells per condition were collected and the corresponding iron content was analyzed by spectrophotometry assay, and finally expressed as pg Fe/cell (*p < 0.05, **p < 0.01 and ***p < 0.001). (B) Pellets of 1.5 × 105 cells per condition were enclosed in agarose phantoms and visualized by a clinical grade 3T scanner using fast field echo T2* sequences. (C) Representative images of Prussian blue staining of DTR/eGFP-transduced BV-2 cells unlabeled and labeled with either ferucarbotran or ferumoxide. (D) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed no differences in cell viability between unlabeled and labeled cells. Data are expressed in cell viability percentages, obtained by normalizing the average values of labeled DTR/eGFP-transduced and control cells for the corresponding average values of unlabeled cells, with each assay performed in triplicate.
(E) The SPIO retention inside BV-2 cells was analyzed by iron quantification at each cell passage, showing that, in all tested labeling conditions, the iron amount completely disappeared after two rounds of cell cycle (6 days, two passages). (E,i) Ferumox. and (E,ii) ferucarb. (A, D & E) The data represent mean ± standard deviation. CTR: Control; DTR: Diphtheria toxin receptor/enhanced green fluorescence protein-transduced; Ferucarb.: Ferucarbotran; Ferumox.: Ferumoxide.
(Figure omitted. See article PDF.)
Figure 2. In vitro evaluation of cell death mediated by diphtheria toxin. (A & B) In order to test the efficiency of DT action in inducing cell death, DTR/eGFP-expressing and control BV-2 cells were incubated with different DT concentrations (ranging from 0.1 to 1000 ng/ml) for 24 h. (A) Cell death analysis (using the Annexin V/7AAD detection kit) and GFP expression evaluation by flow cytometry were performed, showing a strong correlation between the percentages of viable cells and GFP-positive cells. (B) Representative flow cytometry plots of DTR-transduced cells treated with DT (10 ng/ml) are reported. (C & D) To analyze the kinetics of DT action, DTR/eGFP-transduced and control BV-2 cells were incubated with 10 ng/ml of DT and analyzed by flow cytometry (using the Annexin V/7AAD detection kit) and bioluminescence at different time points (0,...